Manipulation of fluids, fluid components and reactions in microfluidic systems

ABSTRACT

Microfluidic structures and methods for manipulating fluids, fluid components, and reactions are provided. In one aspect, such structures and methods can allow production of droplets of a precise volume, which can be stored/maintained at precise regions of the device. In another aspect, microfluidic structures and methods described herein are designed for containing and positioning components in an arrangement such that the components can be manipulated and then tracked even after manipulation. For example, cells may be constrained in an arrangement in microfluidic structures described herein to facilitate tracking during their growth and/or after they multiply.

RELATED APPLICATIONS

This application is a continuation of U.S. nonprovisional patentapplication Ser. No. 14/070,953, filed Nov. 4, 2013, which is acontinuation of U.S. nonprovisional patent application Ser. No.12/595,107, filed May 18, 2010, which is a U.S. national stage filing ofPCT/US08/05009, filed Apr. 18, 2009, which claims priority to and thebenefit of U.S. provisional application Ser. No. 60/925,357, filed Apr.19, 2007, the content of each of which is incorporated by referenceherein in its entirety.

FIELD OF INVENTION

The present invention relates generally to microfluidic structures, andmore specifically, to microfluidic structures and methods formanipulating fluids, fluid components, and reactions.

BACKGROUND

in Microfluidic systems typically involve control of fluid flow throughone or more microchannels. One class of systems includes microfluidic“chips” that include very small fluid channels and smallreaction/analysis chambers. These systems can be used for analyzing verysmall amounts of samples and reagents and can control liquid and gassamples on a small scale. Microfluidic chips have found use in bothresearch and production, and are currently used for applications such asgenetic analysis, chemical diagnostics, drug screening, andenvironmental monitoring. Although these systems may allow manipulationof small volumes of fluids, additional methods that allow furthercontrol and flexibility are needed.

SUMMARY OF THE INVENTION

Microfluidic structures and methods for manipulating fluids, fluidcomponents, and reactions are provided.

In one aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising a first region anda microfluidic channel in fluid communication with the first region,flowing a first fluid in a first direction in the microfluidic channel,and flowing a second fluid in the first direction in the microfluidicchannel. The method also includes partitioning at least a portion of thefirst fluid at the first region, at least in part through action of thesecond fluid, so as to form a first droplet of the first fluid at thefirst region. The method also includes maintaining the droplet at thefirst region while the second fluid is flowing in the first direction.

In another embodiment, a method comprises providing a microfluidicnetwork comprising at least a first inlet to a microfluidic channel, afirst and a second region for forming a first and a second droplet,respectively, the first and second regions in fluid communication withthe microfluidic channel, and flowing a first fluid in the microfluidicchannel. The method involves partitioning a first portion of the firstfluid at the first region, at least in part through action of the secondfluid, so as to form the first droplet at the first region, andpartitioning a second portion of the first fluid at the second region,at least in part through action of the second fluid, so as to form thesecond droplet at the second region.

In another embodiment, a microfluidic device comprises a plurality ofchamber units positioned in parallel, each chamber unit comprising: achamber having a chamber inlet and a chamber outlet, a feed channelfluidly connected to a plurality of chamber inlets, a drain channelfluidly connected to a plurality of chamber outlets, a chamber bypasschannel extending from the chamber, and a fluid restriction regionbetween the chamber outlet and the drain channel, the fluid restrictionregion being more restrictive to fluid flow than the chamber.

In another embodiment, a method comprises flowing a fluid containing aplurality of components in a microfluidic system comprising a chamberhaving a flow direction, a chamber inlet, a chamber outlet, and achamber bypass channel extending from the chamber between the chamberinlet and the chamber outlet. The method also includes positioning acomponent in the chamber, the chamber having a cross-sectional area,perpendicular to the flow direction, less than 2 times the largestcross-sectional area of the component perpendicular to the flowdirection, and flowing a fluid through the chamber while maintaining thecomponent at its position in the chamber. A portion of the plurality ofcomponents may be flowed in the chamber bypass channel.

In another embodiment, a system comprises a microfluidic devicecomprising an inlet, an outlet, a chamber having a flow direction, and aflow restriction region fluidly connected to the outlet of the chamber,and a plurality of cells generally aligned in the chamber. At least 80%of the cells have a largest cross-sectional area, perpendicular to theflow direction, of between 0.1 and 1.0 times the cross-sectional area ofthe chamber perpendicular to the flow direction. The flow restrictionregion is constructed and arranged to allow a fluid but not the cells topass therethrough.

A method may also comprise providing a microfluidic network comprisingat least a first inlet to a microfluidic channel, a first and a secondregion for positioning a first and a second reactive component,respectively, the first and second regions in fluid communication withthe microfluidic channel, wherein the first region is closer in distanceto the first inlet than the second region, and flowing a first fluidcomprising first and second components in the microfluidic channel. Themethod may also include positioning the first component at the firstregion, positioning the second component at the second region, andmaintaining the first and second components in the first and secondregions, respectively, while a fluid is flowing in the microfluidicchannel. In one embodiment, the first and/or second reactive componentis a cell. In another embodiment, the first and/or second reactivecomponent is a bead. In some cases, positioning of the first and/orsecond reactive components does not require use of a fluid immisciblewith the first fluid. The method may optionally include flowing a secondfluid comprising an associating component in the microfluidic channel,wherein the associating component can interact with the first and/orsecond reactive components. The associating component may be a bindingpartner complementary to the first and/or second components. In someembodiments, the microfluidic channel comprises an upstream portion, adownstream portion, and first and second fluid paths extending from theupstream portion and reconnecting at the downstream portion. The firstand second fluid paths may have different resistances to flow. In somecases, the first region is positioned within the first fluid path.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1E show schematically a microfluidic network for positioning adroplet in a region of the network according to one embodiment of theinvention.

FIGS. 2A-2C show schematically removal of a droplet from a region of thenetwork according to one embodiment of the invention.

FIG. 3 is a photograph showing multiple sections of a microfluidicnetwork for positioning droplets according to one embodiment of theinvention.

FIG. 4 is a photograph showing multiple droplets positioned in multipleregions of a microfluidic network according to one embodiment of theinvention.

FIGS. 5A-5C show manipulation of a droplet positioned in a region of amicrofluidic network by changing the surface tension of the dropletaccording to one embodiment of the invention.

FIG. 6 is a photograph of a droplet wetting a surface of themicrofluidic network according to one embodiment of the invention.

FIG. 7 shows de-wetting of the droplet from a surface of themicrofluidic network after being treated with a stabilizing agentaccording to one embodiment of the invention.

FIGS. 8A-8E show a method of forming a droplet and maintaining a dropletat a first region of a microfluidic device according to one embodimentof the invention.

FIG. 9 shows a microfluidic device including several regions for formingdroplets according to one embodiment of the invention.

FIGS. 10A-10F show a method of positioning a reactive component at aregion of a microfluidic device according to one embodiment of theinvention.

FIG. 11 shows a microfluidic device including a plurality of chambersaccording to one embodiment of the invention.

FIGS. 12A-12B show alignment of components in chambers of a microfluidicsystem according to one embodiment of the invention.

FIGS. 13A-13B show an array of single cells positioned in chambers of a,microfluidic system according to one embodiment of the invention.

FIGS. 14A-14B are bright field and fluorescence images, respectively,showing a plurality of cells that have grown from single cells similarto the ones shown in FIGS. 13A and 13B.

FIGS. 15A-15E are photographs showing the growth of cells at a region ofa microfluidic device according to one embodiment of the invention.

FIGS. 16A-16C show fluorescently-labeled cells at a region of amicrofluidic device according to one embodiment of the invention.

FIGS. 17A-17C show cells expressing a fluorescent protein at a region ofa microfluidic device according to one embodiment of the invention.

FIGS. 18A-18B show aligned cells and the tracking of cells in a chamberof a microfluidic system, according to one embodiment of the invention.

FIG. 18C shows a chart of the lineology of the cells shown in FIG. 18B.

FIG. 19 shows aligned cells in a chamber of a microfluidic system. Thecells show different levels of gene expression and therefore havedifferent intensities.

FIGS. 20A-20B show yeast cells grown in a chamber having a plurality ofbranching channels in the form of a grid according to one embodiment ofthe invention.

FIG. 21 shows phenotype switching of cells in response to being exposedto synthetic dextrose media according to one embodiment of theinvention.

FIG. 22 shows FISH staining of cells positioned in a chamber accordingto one embodiment of the invention.

FIGS. 23A-23C show a screen of a GFP library of yeast cells positionedin a chamber according to one embodiment of the invention.

FIGS. 24A-24E show different levels of gene expression in cells that areexposed to various buffers according to one embodiment of the invention.

DETAILED DESCRIPTION

The present invention relates to microfluidic structures and methods formanipulating fluids, fluid components, and reactions. In one aspect,such structures and methods involve positioning fluid samples, e.g., inthe form of droplets, in a carrier fluid (e.g., an oil, which may beimmiscible with the fluid sample) in predetermined regions in amicrofluidic network. In some embodiments, positioning of the dropletscan take place in the order in which they are introduced into themicrofluidic network (e.g., sequentially) without significant physicalcontact between the droplets. Because of the little or no contactbetween the droplets, coalescence between the droplets can be avoided.Accordingly, in such embodiments, surfactants are not required in eitherthe fluid sample or the carrier fluid to prevent coalescence of thedroplets. Positioning of droplets without the use of surfactants isdesirable in certain cases where surfactants may negatively interferewith the contents in the fluid sample (e.g., proteins). Structures andmethods described herein also enable droplets to be removed sequentiallyfrom the predetermined regions to a different region of the fluidicnetwork where they can be further processed.

Once the droplets are positioned at the predetermined regions, they canbe stored and/or may undergo manipulation (e.g.; diffusion, evaporation,dilution, and precipitation). In some instances, many (e.g., 1000)droplets can be manipulated, sometimes simultaneously. Manipulation offluid samples can be useful for a variety of applications, includingtesting for reaction conditions, e.g., in crystallization, and chemicaland/or biological assays, including chemical reactions, enzymaticreactions, immuno-based reactions (e.g., antigen-antibody), andcell-based reactions.

It should be understood that while several of the embodiments describedherein refer to the positioning and/or manipulation of droplets, theembodiments are also applicable to other components such as cells andbeads, which may be contained in a fluid without being in a droplet.

In another aspect, microfluidic structures and methods described hereincan allow production of droplets of a precise volume, which can bestored/maintained at precise regions of the device. The droplets can becreated at a region (e.g., a storage region) in a self-regulated manner.The method may include, optionally, filling a microfluidic channel witha filling fluid (e.g., oil). The oil can then be flushed out with afirst fluid (e.g., an aqueous fluid) to be stored/maintained at a regionof the device. This first fluid may be immiscible with the fillingfluid. The first fluid can enter a region of the device for storing adroplet, replacing the filling fluid in that region. Next, a secondfluid (e.g., a fluid immiscible with the first fluid) may be flowed inthe channel, causing partitioning of a portion of the first fluid.Partitioning of the first fluid causes formation of a droplet of thefirst fluid at the region, while a second portion of the first fluidbypasses the region. In this manner, a plurality of droplets can begenerated sequentially down the length of the channel.

In another aspect, microfluidic structures and methods described hereinare designed for containing and positioning components in an arrangementsuch that the components can be manipulated and then tracked even aftermanipulation. For example, cells may be constrained in an arrangement inmicrofluidic structures described herein to facilitate tracking duringtheir growth and/or after they multiply. This can allow, for example: 1)cells to be trapped and observed over time; 2) culturing of cells in amanner than allows determination of their identity and lineage; and 3)manipulation of the cells (e.g., by staining or washing) whilemaintaining the identity and/or position of the cells. Other advantagesand applications are described in more detail below.

Certain microfluidic chips described herein may include a microfluidicnetwork having a region for forming droplets of sample in a carrierfluid (e.g., an oil), and one or more regions (e.g., microreactorregions, microwells, reservoirs, chambers, or portions of a microfluidicchannel) in which the droplets can be positioned and reaction conditionswithin the droplet can be varied. In some embodiments, the dropletformation region is the same as the region in which the droplet ispositioned for varying a condition within the droplet. Droplets may bepositioned sequentially in regions of the microfluidic network so thatupon manipulating and/or performing a chemical and/or biological processwithin each the droplets, the droplets can be identified at a latertime, for example, to determine the particular conditions within thedroplets that lead to a favorable outcome (e.g., optimal conditions forforming a product, for crystal growth, etc.).

As used herein, “droplet” means a small portion of a fluid, isolatedfrom other portions of the same fluid. A droplet can have a traditional,rounded shape, or a different shape which can be influenced by itsenvironment. A droplet of a first fluid can be surrounded by different,immiscible fluid, or bounded by a surface of an article, or a gas suchas air, or a combination. For example, a droplet of a first fluid can besuspended in (completely surrounded by) a second fluid immiscible withthe first fluid. Or a droplet of a first fluid can reside on a surfaceof a solid article, with portions that are not in contact with thesurface exposed to the second fluid or a gas. A droplet can be boundedon multiple sides by one or more surfaces of an article, e.g. theinterior of a channel. For example, a portion of a channel completelyfilled with a first fluid, which resides within a discrete regions ofthe channel, is a droplet for purposes of the invention.

It should also be understood that any suitable fluid(s) can be used inconnection with devices and methods described herein. Where embodimentsdescribe the use of “immiscible” fluids, those of ordinary skill in theart know or can determine by simple experimentation which combination offluids is immiscible. For instance, solubility parameters of a varietyof fluids are available in literature and can be used to determinemiscibility/immiscibility. Additionally and/or alternatively, simpleexperimentation may include, for example, mixing two or more fluids in acontainer—if the fluids partition after a certain period of time, thefluids are immiscible. Furthermore, it should be understood that where“first” and “second” fluids are described herein, these fluids can haveany suitable composition and can be interchangeable in otherembodiments. For example, one particular embodiment may describe the useof a “first fluid” that is aqueous and a “second fluid” that is an oil,and a different embodiment may described a “first fluid” as an oil and a“second fluid” that is aqueous. In certain embodiments, first and secondfluids can be miscible with one another (e.g., both being aqueous orboth being an oil). Gaseous fluids may also be used.

FIG. 1 shows a method for positioning a droplet in a region of amicrofluidic network according to one embodiment of the invention. Asshown in illustrative embodiment of FIG. 1A, microfluidic network 1000comprises section 1001 including microfluidic channel 1002 having anupstream portion 1006 and a downstream portion 1010 (as fluid flows inthe direction of arrow 1012), with fluid path 1014 and fluid path 1018(e.g., a bypass channel) extending from the upstream portion andreconnecting at the downstream portion. In some cases, resistance tofluid flow (hydrodynamic resistance) may differ between fluid paths 1014and 1018. For example, fluid path 1014 may have less resistance to fluidflowing in the direction of arrow 1012 prior to positioning of a dropletin this section of the microfluidic network. As shown in thisillustrative embodiment, fluid path 1014 has a lower resistance to fluidflow than fluid path 1018 due to the relatively longer channel length offluid path 1018. It should be understood, however, that the microfluidicnetwork may have other designs and/or configurations for impartingdifferent relative resistances to fluid flow, and such designs andconfigurations can be determined by those of ordinary skill in the art.For instance, in some embodiments, the length, width, height, and/orshape of the fluid path can be designed to cause one fluid path to havea resistance to fluid flow different from another fluid path. In otherembodiments, at least a portion of a fluid path may include anobstruction such as a valve (which may change hydrodynamic resistancedynamically), a semi-permeable plug (e.g., a hydrogel), a membrane, oranother structure that can impart and/or change resistance to fluid flowthrough that portion.

As shown in FIGS. 1B and 1C, droplet 1020 flows in the direction of1012, e.g., by being carried by a carrier fluid 1021 flowing in the samedirection. Upon passing the junction between flow paths 1014 and 1018 atupstream portion 1006, the droplet flows in fluid path 1014 due to itslower resistance to flow in that fluid path relative to fluid path 1018.However, as fluid path 1014 includes a fluid restriction region 1024(e.g., a “narrow fluid path portion” and/or a region having a smallercross-sectional area than that of fluid path portion 1014), droplet 1020cannot flow further down the microfluidic network. Accordingly, droplet1020 is positioned within a region 1028 (e.g., a “microwell” or“chamber”) of the microfluidic network. In some embodiments, droplet1020 can be maintained at the region even though carrier fluid continuesto flow in the microfluidic network (e.g., in the direction of arrow1012).

It should be understood that any suitable fluid path can be used as afluid restriction region, which may have a higher hydrodynamicresistance and/or a smaller cross-sectional area for fluid flow than aregion immediately upstream or downstream of the fluid constrictionregion. For instance, fluid restriction region 1024 may be in the formof a narrow fluid path or a channel having the same dimensions as fluidpath 1014, but having an obstruction (e.g., posts or a valve) positionedin or at the region. In other embodiments, fluid restriction region 1024may comprise a porous membrane, a semi-permeable plug (e.g., a gel), avalve, or another structure.

As shown in the embodiment illustrated in FIG. 1D, the positioning ofdroplet 1020 at region 1028 causes fluid path 1014 to be plugged suchthat no or minimal fluid flows past fluid restriction region 1024. Thisplugging of fluid path 1014 causes a higher resistance to fluid flow inthat path compared to that of fluid path 1018. As a result, when asecond droplet 1030 flows in the direction of arrow 1012, the seconddroplet bypasses flow path 1014 and enters flow path 1018, which now hasa lower hydrodynamic resistance than that of fluid path 1014 (FIG. 1D).Accordingly, second droplet 1030 can bypass first droplet 1020 and cannow be positioned in a second region within microfluidic network 1000(not shown).

It should be understood that when droplet 1020 is positioned at region1028, the droplet may plug all or a portion of fluid path 1014 and/orfluid restriction region 1024. For instance, in some cases, the dropletplugs all of such fluid paths such that none of carrier fluid 1021 (oranother fluid) flowing in microfluidic channel 1002 passes through fluidrestriction region 1024. In other embodiments, the droplet may plug onlya portion of such fluid paths such that some fluid passes through fluidrestriction region 1024 even though the droplet is positioned at region1028. The amount of fluid flowing past the positioned droplet may dependon factors such as the dimensions of fluid path portions 1014 and/or1024, the size of the droplets, the flow rate, etc. As the dropletcauses fluid path 1014 to have a higher relative hydrodynamic resistancethan fluid path 1018, a second droplet can bypass fluid path 1014 andenter fluid path 1018.

As described above, fluid paths 1014 and 1018 may have differenthydrodynamic resistances depending on whether or not a droplet ispositioned at region 1028. In the absence of a droplet positioned atregion 1028, fluid path 1014 may be configured to have a lowerhydrodynamic resistance than fluid path 1018. For example, greater than50%, greater than 60%, greater than 70%, greater than 80%, or greaterthan 90% of the fluid flowing in channel 1002 at upstream portion 1006may flow in fluid path 1014 compared to fluid path 1018. However, whenthe droplet is positioned and maintained in region 1028, fluid path 1014may be relatively more restrictive to fluid flow. For example, less than50%, less than 40%, less than 30%, less than 20%, or less than 10% ofthe fluid flowing in channel 1002 at upstream portion 1006 may flow influid path 1014 compared to that of 1018. In some cases, 100% of thefluid flowing in direction 1012 in microfluidic channel 1002 flows influid path 1018 upon positioning of a droplet in region 1028.

As illustrated in the exemplary embodiment of FIG. 1, the positioning ofdroplet 1020 (e.g., a first droplet) and the subsequent bypass ofdroplet 1030 (e.g., a second droplet) does not require contact betweenthe first and second droplets due to the design of section 1001. Incertain embodiments, the second droplet does not physically contact thefirst droplet after positioning of the first droplet in region 1028.This can occur, in some embodiments, when the volume and/or length offluid path 1014 (between fluid restriction region 1024 and theintersection between fluid paths 1014 and 118) is larger than the volumeand/or length of droplet 1020. In other embodiments, the second dropletcan come into physical contact with the first droplet as it bypasses thefirst droplet, however, due to such minimal contact between the twodroplets, the droplets do not coalesce. This can occur, in someembodiments, when the volume and/or length of fluid path 1014 (betweenfluid restriction region 1024 and the intersection between fluid paths1014 and 118) is smaller than the volume and/or length of droplet 1020.

Accordingly, in some instances, the positioning of the droplets in themicrofluidic network can take place without the use of surfactants. Inother words, surfactants in either a fluid flowing in channel 1002(e.g., a carrier fluid) or within the droplets is not required in orderto stabilize the droplets and/or prevent the droplets from coalescingwith one another during positioning or carrying the droplet in themicrofluidic channel, and/or during maintaining the droplets within apredetermined region within the microfluidic network. However, ininstances where coalescence is desired (e.g., to allow a reactionbetween reagents contained in two droplets), the microfluidic networkand methods for operating the network can be configured to allow suchphysical contact and/or coalescence between droplets. These interactionsor absence of interactions can be controlled, for example, by varyingthe volume and/or length of the droplets, as well as the volume and/orlength of regions 1028.

In some embodiments, methods for positioning a droplet in a microfluidicnetwork include the steps of providing a microfluidic network comprisinga first region (e.g., region 1028 of FIG. 1A) and a microfluidic channelin fluid communication with the first region, flowing a first fluid(e.g., a carrier fluid) in the microfluidic channel, and flowing a firstdroplet comprising a second fluid (e.g., a fluid sample) in themicrofluidic channel, wherein the first fluid and the second fluid areimmiscible. The first droplet may be positioned in the first region andmaintained in the first region while the first fluid is flowing in themicrofluidic channel. In such embodiments, positioning and/ormaintaining the first droplet in the first region does not require theuse of a surfactant in the first or second fluids. As described in moredetail below, other components such as cells and beads may be positionedin addition to or instead of droplets in a similar manner.

In some embodiments, a chemical and/or biological process and/or anothermanipulation process can be carried out in droplet 1020 of FIG. 1 whilethe droplet is positioned in region 1028. For example, a fluid sample inthe droplet may undergo a process such as diffusion, evaporation,dilution, and/or precipitation. The droplet may be manipulation, forexample, by changing the concentration of the fluid flowing in channel1002 after the droplet has been positioned at region 1028. In otherembodiments, region 1028 is in fluid communication with another fluidicchannel, flow path, reservoir, or other structure, e.g., via a semipermeable membrane that may be positioned adjacent the region (e.g.,underneath or above region 1028), and manipulation of the droplet canoccur via such passages. Manipulations of fluids are described in moredetail in U.S. Application Ser. No. 60/925,357, filed Apr. 19, 2007, andentitled “Manipulation of Fluids and Reactions in Microfluidic Systems”,which is incorporated herein by reference in its entirety for allpurposes.

In some embodiments, droplets that have been positioned at regions of amicrofluidic network can be removed or extracted from the regions to adifferent location in the microfluidic network, where they can beoptionally processed, manipulated, and/or collected. As shown in theillustrative embodiments of FIGS. 2A-2C, removing droplet 1020 fromregion 1028 of section 1001 of microfluidic network 1000 can take placeby reversing the flow of the carrier fluid in the network such that thecarrier fluid now flows in the direction of arrow 1040 (instead of inthe direction of arrow 1012 of FIGS. 1A-1E).

In some such embodiments, upstream portion 1006 and downstream portion1010 of FIGS. 1A-1E now become reversed such that portion 1010 is now anupstream portion and portion 1006 is now a downstream portion. The flowof a carrier fluid in the direction of arrow 1040 in microfluidicchannel 1002 causes a portion of the fluid to flow through fluidrestriction region 1024 into region 1028 where droplet 1020 ispositioned. This fluid flow causes the droplet to flow in the directionof arrow 1040. As shown in the embodiment illustrated in FIG. 2B,droplet 1030, which may have been positioned at a different region ofthe microfluidic network, can be removed from that region and may alsoflow in the direction of arrow 1040. As droplet 1030 encounters fluidrestriction region 1024, the droplet cannot flow through this narrowopening due to the region's high hydrodynamic resistance. As a result,the droplet bypasses fluid restriction region 1024 and flows into fluidpath 1018 until it reaches microfluidic channel 1002 at downstreamportion 1006. Thus, by reversing the flow and the pressure gradient inthe microfluidic network, droplets 1020 and 1030 can be removedsequentially from the regions of the microfluidic network where theypreviously resided. That is, droplet 1020, which was positioned firstbefore droplet 1030, can be removed from its region and can enter adifferent region of the microfluidic network before that of droplet1020. Optionally, when droplet 1030 reaches downstream portion 1006, theflow can be reversed again (e.g., such that fluid flows in the directionof arrow 1012 of FIGS. 1A-1E) to cause droplet 1030 to enter into region1028. This method can allow droplets to be removed from a first regionand positioned in a second region of the microfluidic network.

In some embodiments, sequential positioning of droplets can be performedsuch that a first droplet is positioned in a first region before asecond droplet is positioned in a second region (and, optionally, beforethird, fourth, fifth droplets, etc. are positioned in their respectiveregions). As described above, sequential removal of the droplets can beperformed such that the first droplet is removed from a region and/orpositioned at a different location of the microfluidic network beforethe second droplet (and, optionally, before third, fourth, fifthdroplets, etc. are removed from their respective regions). In otherembodiments, removal of the droplets can be performed such that thesecond droplet is removed and/or positioned at a different location ofthe microfluidic network before the first droplet.

In some cases, several (e.g., greater than 2, greater than 5, greaterthan 10, greater than 50, greater than 100, greater than 200, greaterthan 500, or greater than 1000) droplets can be positioned at regions ofthe microfluidic network, wherein the droplets are positioned in theregions in the order the droplets are introduced into the microfluidicnetwork. In some cases, removing several droplets positioned at regionsof the microfluidic network comprises removing the droplets in the orderthe droplets were introduced into the microfluidic network (or in theorder the droplets were positioned into the regions of the microfluidicnetwork). In other cases, removing several droplets positioned atregions of the microfluidic network comprises removing the droplets inthe reverse order the droplets were introduced into the microfluidicnetwork (or in the reverse order the droplets were positioned into theregions of the microfluidic network). Other methods of positioning andremoval of droplets are also possible.

The sequential (or predetermined/known order of) removal of dropletsfrom regions of a microfluidic network can allow control over theidentification and location of each droplet within the network. This canalso allow determination of the contents inside each of the dropletsfrom the time they are formed and/or introduced into the microfluidicnetwork, to the time the droplets are manipulated and/or extracted fromthe microfluidic network.

FIG. 3 is a photograph of multiple sections 1001-A, 1001-B, and 1001-Cof microfluidic network 1050 according to one embodiment of theinvention. A carrier fluid may flow in microfluidic channel 1002-A inthe direction of arrow 1012 from an inlet positioned upstream of portion1006. The carrier fluid may partition at the junction where fluid paths1014-A and 1018-A extend from microfluidic channel 1002. The proportionof fluid that flows in each of the fluid paths can be determined atleast in part by the relative hydrodynamic resistances of the paths, asdescribed above. In the embodiment shown in FIG. 3, sections 1001-A,1001-B, and 1001-C are positioned in series. In other embodiments,however, such sections may be positioned in parallel and/or in bothseries and parallel. Other configurations are also possible.

A microfluidic network may have any suitable number of microfluidicsections 1001. For instance, the microfluidic network may have greaterthan or equal to 5, greater than or equal to 10, greater than or equalto 30, greater than or equal to 70, greater than or equal to 100,greater than or equal to 200, greater than or equal to 500, or greaterthan or equal to 1000 such sections.

In additional, although certain embodiments herein show that sections1001 can allow positioning of a single droplet in each of the sections,in other embodiments, the sections can be designed such that greaterthan one droplet (e.g., greater than or equal to 2, greater than orequal to 5, or greater than or equal to 10 droplets) can be positionedat each section.

Furthermore, although only two fluid flow paths 1014 and 1018 are shownextending from channel 1002, in other embodiments, more than two (e.g.,greater than or equal to 3, greater than or equal to 5, or greater thanor equal to 10) fluid paths may extend from channel 1002. Each extendingfluid path may optionally comprise one or more regions (e.g.,microwells) for positioning and/or maintaining droplets.

FIG. 4 shows the positioning of droplets 1060, 1062, and 1064 atpositions 1028-A, 1028-B, and 1028-C, respectively, in microfluidicnetwork 1050 according to one embodiment of the invention. As shown inthis illustrative embodiment, carrier fluid 1021 flows in the directionof arrow 1012 and carries droplet 1060 through channel 1002-A and intofluidic path 1014-A due to the lower resistance to fluid flow in thatfluid path compared to that of fluid path 1018-A. That is, prior to thepositioning of droplet 1060 in region 1028-A, more than 50% of the fluidflowing in microfluidic channel 1002-A flows through fluid path 1014-Acompared to fluid path 1018-A.

Once droplet 1060 is positioned at region 1028-A, it impedes fluid flowthrough fluid restriction region 1024-A such that the hydrodynamicresistances of fluid paths 1014-A and 1018-A are altered. This causesthe hydrodynamic resistance of portion 1014-A to be higher, and as aresult, a greater amount of fluid flows in the direction of 1070 throughfluid path portion 1018-A. Accordingly, a second droplet 1062 flowingthrough microfluidic channel 1002-A and passing upstream portion 1006now bypasses fluid path portion 1014-A and flows through portion 1018-A.The second droplet, after bypassing region 1028-A, now entersmicrofluidic channel portion 1002-B. If there is a lower hydrodynamicresistance in fluid path portion 1014-B compared to region 1018-B (e.g.,a droplet has not already been positioned in region 1028-B), the dropletcan be positioned at this region. Next, a third droplet 1064 can flowthrough microfluidic channel portion 1002-A in the direction of arrow1012 and first bypasses region 1028-A due to droplet 1060 alreadypositioned at that region. The droplet can then flow into fluid pathportion 1018-A and 1002-B. Since droplet 1062 has already beenpositioned at region 1028-B, third droplet 1064 bypasses this region andtakes the fluid path of least hydrodynamic resistance (fluid pathportion 1018-B). Upon entering an empty region such as region 1028-C,the third droplet can now be positioned at that region due to a lowerhydrodynamic resistance in fluid path 1014-C compared to that of fluidpath portion 1018-C (e.g., prior to any other droplet being positionedat region 1028-C).

Accordingly, a method for positioning droplets in regions of amicrofluidic network may include providing a microfluidic networkcomprising at least a first inlet to a microfluidic channel (e.g.,positioned upstream of portion 1006 of FIG. 4), a first region (e.g.,region 1028-A) and a second region (e.g., region 1028-B) for positioninga first and a second droplet, respectively, the first and second regionsin fluid communication with the microfluidic channel, wherein the firstregion is closer in distance to the first inlet than the second region.The method can include flowing a first fluid (e.g., a carrier fluid) inthe microfluidic channel, flowing a first droplet (e.g., a first fluidsample), defined by a fluid immiscible with the first fluid, in themicrofluidic channel, and positioning the first droplet in the firstregion. The method can also include flowing a second droplet (e.g., asecond fluid sample), defined by a fluid immiscible with the firstfluid, in the microfluidic channel past the first region without thesecond droplet physically contacting the first droplet. The seconddroplet may then be positioned at the second region. In some instances,the first and/or second droplets are maintained at their respectiveregions while fluid continues to flow in the microfluidic channel.

It should be understood that other components may be integrated withfluidic networks described herein in some embodiments of the invention.For example, in some instances, hydrodynamic resistances of fluid pathscan be changed dynamically such that the direction of fluid flow (and,therefore, positioning of droplets) can be controlled by the user. Inone such embodiment, valves may be positioned at one or more ofpositions 1070-A, 1070-B, and 1070-C of FIG. 4. For example, a valve atposition 1070-B can cause restriction of fluid flow through fluid pathportion 1014-B, e.g., prior to a droplet being positioned at region1028-B. This can cause a droplet flowing through microfluidic channelportion 1002-B to bypass region 1028-B even though a droplet is notpositioned at that region. Thus, the droplet flowing through portion1002-B will flow through fluid path 1018-B and onto the next availableregion, where the fluid resistance of that region may or may not becontrolled by a similar valve. In some instances, after a dropletbypasses region 1028-B due to a closed valve at position 1070-B (or anyother component that can change the relative resistances to fluid flowbetween fluid paths 1014-B and 1018-B), the valve at position 1070-B cannow be reopened to change the relative resistances to fluid flow suchthat a next droplet can now enter into region 1028-B and be positionedat that region. Such a system can allow droplets to be positioned at anydesired region of a microfluidic network.

As described herein, in some embodiments droplets do not requirestabilization (e.g., the use of surfactants or other stabilizing agents)in order to be positioned at predetermined regions within microfluidicnetworks described herein. This is because in some embodiments, thedroplets do not significantly physically contact one another duringbypass of one droplet to another. Due to the little or no physicalcontact between the droplets, the droplets do not have a chance tocoalesce with one another. Thus, surfactants or other stabilizing agentsare not required to stabilize the droplets from coalescing in some suchembodiments.

In some embodiments, the absence of surfactants or other stabilizingagents causes the droplets to wet a surface of the microfluidic network.Even though wetting may occur, the droplets can still be positioned atpredetermined regions within the microfluidic network due to, forexample, a positive pressure that causes fluid flow to carry thesedroplets into these regions. As discussed above, the use of dropletsand/or a carrier fluid that does not contain a surfactant isadvantageous in some embodiments where surfactants may negativelyinterfere with contents inside the droplets. For example, the dropletsmay contain proteins, and surfactants are known to denature certainproteins to some extent. However, after manipulation of the dropletand/or carrying out a process such as a chemical and/or biologicalreaction inside the droplet, surfactants may no longer negatively affectthe contents inside the droplet. Accordingly, in such cases, asurfactant or other stabilizing agent can be applied to the dropletsafter the droplets have been positioned at regions of the microfluidicnetwork. In some embodiments, application of a stabilizing agent to adroplet after manipulation of the droplet and/or carrying out a processinside the droplet can facilitate mobilization of the droplet out of theregion in which the droplet is positioned.

It should be understood, however, than in some embodiments, a dropletand/or a carrier fluid may contain a surfactant or other stabilizingagent that stabilizes a droplet prior to positioning of the droplet at aregion in the microfluidic network. In some such embodiments, thestabilizing agent does not negatively interfere with contents (e.g.,reagents) inside the droplet. Of course, such embodiments will depend ona variety of factors such as the type of stabilizing agent used, thecontents inside the droplet, the application, etc.

FIGS. 5A-5C show schematically the treatment of a droplet positioned ata predetermined region within a microfluidic network with a stabilizingagent according to one embodiment of the invention. As described above,droplet 1080 (which, in this embodiment, does not include a stabilizingagent) can be positioned at region 1028 by flowing a carrier fluid andthe droplet in the direction of arrow 1012. After the droplet has beenpositioned, the droplet may wet a surface of the channel, such assurface portions 1084. (In other cases, however, the surface of thechannel can be treated with a chemical coating so that the droplet doesnot wet the surface of the channel.) In some embodiments, wetting of thechannel surface can cause the droplet to be immobilized at this region,even when a carrier fluid is flowed in the opposite direction (e.g., inthe direction of arrow 1088) in attempt to remove the droplet from thisregion. In some such embodiments, a fluid comprising a stabilizing agent(e.g., a surfactant) can be flowed in the microfluidic network, e.g., inthe direction of arrow 1088 through microfluidic channel 1002. A portionof this fluid can flow through fluid restriction region 1024 to reachdroplet 1080 at region 1028. This fluid containing the stabilizing agentcan cause the droplet to be coated with the stabilizing agent, which canresult in the droplet de-wetting from the channel at surface portions1084. In such cases, the surface tension of the droplet has beenreduced. Thus, the droplet may be “depinned” from one or more surfacesof the channel.

If desired, after introducing a fluid containing a stabilizing agent tothe droplet, the fluid flow may be stopped for a certain amount of timeto allow the stabilizing agent to coat the droplet. In otherembodiments, however, flow in channel 1002 is not stopped after thestabilizing agent has been introduced. In yet other embodiments, after adroplet has been de-wetted from a surface of the microfluidic network,fluid flowing in the microfluidic network may be replaced by a secondfluid (which may or may not contain a stabilizing agent). As shown inthe embodiment illustrated in FIG. 5C, droplet 1080 can beremoved/extracted from region 1028 in the direction of arrow 1088. Oneof ordinary skill in the art can determine appropriate conditions forde-wetting a droplet from a surface of the microfluidic network whichmay depend on conditions such as the concentration of the stabilizingagent in the fluid, the flow rate, the degree of wetting of the droplet,the contents of the droplet, the material composition of the fluidicnetwork, as well as other factors.

FIG. 6 is a photograph showing droplet 1092 that has wetted surfaceportions 1096 of microfluidic network 1090 at region 1028. As shown inFIG. 7, after flowing a fluid containing a surfactant in the directionof arrow 1088, a portion of which flows through a fluid restrictionregion 1024, droplet 1092 de-wets surface portions 1096 and is nowstabilized with the stabilizing agent. The stabilization is evident bymeniscus 1098 that forms around droplet 1092, as the droplet now has alower energy state configuration compared to that shown in FIG. 6.

It should be understood that a fluid containing a stabilizing agent canbe introduced into microfluidic network 1090 in any suitable manner. Forexample, in some embodiments, the stabilizing agent may be introduced bya fluid flowing in the direction of arrow 1012. In other embodiments,region 1028 may be in fluidic communication with another portion of thedevice extending from region 1028. For instance, above or below region1028 may be a reservoir, a channel, or other component that can be usedto introduce a stabilizing agent or other entity to a droplet in thatregion.

As shown in FIGS. 2 and 5, droplets that are released from a region of amicrofluidic network can be forced to flow in a direction opposite thatwhich was used to position the droplet in the region. In otherembodiments, however, after a droplet has been removed from region inwhich it was positioned, the droplet may be forced to flow in the samedirection as that which was used to position the droplet. For example,in one embodiment, droplet 1080 of FIG. 5C can be released from position1028 and can be forced to flow in the direction of 1088 until thedroplet resides at a downstream portion of channel 1002 (e.g., at thetop of microfluidic network 1000 as shown in FIG. 5C). Then, a valve orother component that may be positioned at position 1070 can be at leastpartially closed to cause a higher resistance to fluid flow in fluidflow path 1014 compared to that of 1018. Since fluid flow path 1018 nowhas a lower resistance to fluid flow, flow of the carrier fluid can nowbe reversed such that it flows in the direction of arrow 1012, in whichcase the droplet can bypass fluid flow path 1014 and enter fluid flowpath 1018.

Different types of carrier fluids can be used to carry droplets orcomponents in a microfluidic system. Carrier fluids can be hydrophilic(e.g., aqueous) or hydrophobic (e.g., an oil), and may be chosendepending on the type of droplet being formed or positioned (e.g.,aqueous or oil-based) and/or the type of process occurring in thedroplet. (e.g., crystallization or a chemical reaction). In some cases,a carrier fluid may comprise a fluorocarbon. In some embodiments, thecarrier fluid is immiscible with the fluid in the droplet. In otherembodiments, the carrier fluid is slightly miscible with the fluid inthe droplet. Sometimes, a hydrophobic carrier fluid, which is immisciblewith the aqueous fluid defining the droplet, is slightly water soluble.For example, oils such as PDMS and poly(trifluoropropylmethysiloxane)are slightly water soluble. These carrier fluids may be suitable, forexample, when fluid communication between the droplet and another fluidis desired. Diffusion of water from a droplet, through the carrierfluid, and into a second droplet is one example of such a case.

As described above, methods for storing and/or extracting droplets in amicrofluidic network are provided herein. In some embodiments, thedroplets may be stored and/or extracted in sequential order. Forexample, the droplets may be extracted in the order they are stored orpositioned in predetermined regions in the microfluidic network. Inother embodiments, the use of valves can allow only certain droplets tobe released from regions of the microfluidic system. Advantageously, insome embodiments, such methods do not require the use of surfactants orother stabilizing agents, since the droplets may not come intosubstantial physical contact with one another in a manner that causescoalescence. This is advantageous in certain cases as surfactants mayinterfere with contents such as proteins inside the droplet, as is knownto those of ordinary skill in the art.

In another aspect of the invention, a method of forming droplets inregions of a microfluidic device is provided. The method can allowproduction of droplets of a precise volume, which can bestored/maintained at precise regions of the device. The drops can becreated at the storage region in a self-regulated manner. The method mayinclude, optionally, filling a microfluidic channel with a filling fluid(e.g., oil). The oil can then be flushed out with a first fluid (e.g.,an aqueous fluid) to be stored/maintained at a region of the device.This first fluid may be immiscible with the filling fluid. The firstfluid can enter a region of the device for storing a droplet, replacingthe filling fluid in that region. Next, a second fluid (e.g., a fluidimmiscible with the first fluid) may be flowed in the channel, causingpartitioning of a portion of the first fluid (e.g., at least in partthrough action of the second fluid). Partitioning of the first fluidcauses formation of a droplet of the first fluid at the region, while asecond portion of the first fluid bypasses the region. In this manner, aplurality of droplets can be generated sequentially down the length ofthe channel.

Advantageously, the devices and methods described herein may addressseveral problems commonly associated with forming and/or storingdroplets: (1) Each droplet of the same volume may be formed (or, thedroplets may have different volumes, e.g., depending on the size of thepositioning regions) and all of the first fluid (e.g., aqueous phase)may be used with zero or minimal waste, (2) Because the forming and/orpositioning of the droplets is done by the serial application of singlephase fluids, the process is simple and tolerant to a wide range of flowrates, pressures, fluids, and materials used to form the device. (3)Valves are not required in this device, which may make it easy tomanufacture (although valves may be used with the device if desired),(4) Drop generation is robust and simple (e.g., in contrast to certainflow focusing and T-junction methods), (5) The method can be used toposition/store reactive components besides (or in addition to) droplets,such cells as beads for use in PCR and ELISA type bioassays.

FIG. 8 shows a method for forming, positioning, and/or maintaining adroplet in a region of a microfluidic network according to oneembodiment of the invention. The device used in this method may have thesame configuration as that described in connection with FIGS. 1-7. Asshown in illustrative embodiment of FIG. 8A, microfluidic network 1000comprises section 1001 including microfluidic channel 1002 having anupstream portion 1006 and a downstream portion 1010 (as fluid flows inthe direction of arrow 1012), with fluid paths 1014 and 1018 extendingfrom the upstream portion and reconnecting at the downstream portion. Insome cases, resistance to fluid flow may differ between fluid paths 1014and 1018. For example, fluid path 1014 may have less resistance to fluidflowing in the direction of arrow 1012 prior to forming, positioning,and/or maintaining of a droplet in this section of the microfluidicnetwork. As shown in this illustrative embodiment, fluid path 1014 has alower hydrodynamic resistance than fluid path 1018 due to the relativelylonger channel length of fluid path 1018. It should be understood,however, that the microfluidic network may have other designs and/orconfigurations for imparting different hydrodynamic resistances, andsuch designs and configurations can be determined by those of ordinaryskill in the art. For instance, in some embodiments, the length, width,height, and/or shape of the fluid path can be designed to cause onefluid path to have a resistance to fluid flow different from anotherfluid path. In one particular embodiment, fluid path 1018 has at leastone cross-sectional dimension (e.g., a width or height) that is lessthan a cross-sectional dimension of fluid path 1014. In otherembodiments, at least a portion of a fluid path may include anobstruction such as a valve (which may change hydrodynamic resistancedynamically), a semi-permeable plug (e.g., a hydrogel), a membrane, oranother structure that can impart and/or change hydrodynamic resistancein that portion.

FIG. 8A shows an empty microfluidic channel. In one embodiment, afilling fluid 1011 (e.g., an oil) is flowed into channel 1002, fillingthe channel and fluid paths 1014 and 1018 as shown in FIG. 8B. As shownin FIG. 8C, a first fluid 1200 (e.g., a fluid to be stored as droplets)is flowed into channel 1002 in the direction of arrow 1012. At upstreamportion 1006, a first portion of the fluid flows into fluid path 1014while a second portion of the fluid flows into fluid path 1018. (Thefirst fluid may flow into fluid path 1014 before fluid path 1018 iffluid path 1014 is designed to have a lower hydrodynamic resistance thanfluid path 1018.) As shown in the embodiment illustrated in FIG. 8D,once the first portion of the first fluid reaches a fluid restrictionregion 1024, the first portion cannot pass through this narrow portiondue to the high hydrodynamic resistance of this fluid path (e.g., ameniscus formed between the first fluid and the filling fluid may cause“plugging” of the narrow fluid path). In some embodiments, the fillingfluid can pass through fluid restriction region 1024, although in otherembodiments, there is little or no fluid flow through this region.

As shown in FIG. 8E, a second fluid 1202 may then be flowed into channel1002 in the direction of arrow 1012. This second fluid may be immisciblewith the first fluid. At upstream portion 1006, the second fluidbypasses fluid path 1014 (including region 1028) due the presence of thefirst fluid at that region. The flowing of this second fluid causespartitioning of the first fluid so as to form droplet 1200-A at thefirst region. Fluid path 1018, now having a lower resistance to fluidflow, allows second portion 1200-B of the first fluid to continueflowing in the direction of arrows 1013 and 1015, followed by secondfluid 1202. As fluid path 1014 includes fluid restriction region 1024,droplet 1200-A cannot flow further down the microfluidic network.Accordingly, droplet 1200-A is positioned within a region 1028 (e.g., a“microwell”) of the microfluidic network. In some embodiments, droplet1200-A can be maintained at the region even though fluid continues toflow in the microfluidic network (e.g., in the direction of arrow 1012).

The size/volume of droplet 1200-A can vary and may depend and can bedetermined, at least in part, on the size/volume of region 1028. In someinstances, droplets formed at a region have the same volume (or length)as that of the region. In other instances, droplets formed at a regionhave a different (e.g., a smaller or larger) volume (or length) as thatof the region. The droplet may have a volume (or length) that is within,for example, 5%, 10%, 15%, 20%, 25%, or 30% of the volume (or length) ofthe region in which the droplet is positioned. In other cases, thevolume (or length) of a droplet formed in a region is substantiallysmaller (e.g., less than 50%) than the volume (or length) of the region.For instance, if a region having a volume (or length) of X alreadycontains a droplet having a volume (or length) of Y (e.g., using amethod of positioning droplets as described in connection with FIGS.1-7), a second droplet having an approximate volume (or length) of X−Y(X minus Y) may be formed/stored at the region by the methods describedin connection with FIG. 8. This can allow the formation and/orpositioning of multiple droplets in a single region of the device.Various sizes/volumes of droplets that can be formed/stored aredescribed in more detail below.

As described above, in some embodiments, the positioning/presence ofdroplet 1200-A at region 1028 of FIG. 8 causes fluid path 1014 to beplugged such that no or minimal fluid flows past fluid restrictionregion 1024. This plugging of fluid path 1014 causes it to have a higherhydrodynamic resistance compared to fluid path 1018. As a result, whensubsequent fluids are flowed in the direction of arrow 1012, thesubsequent fluids bypass flow path 1014 and enter flow path 1018, whichnow has a lower resistance than that of fluid path 1014. Accordingly,once region 1028 has been “filled” with a droplet, other regionsdownstream can be filled with droplets (e.g., by the formation ofdroplets at these regions). This allows a plurality of droplets to begenerated/stored sequentially down the length of the channel.

Accordingly, one method of the invention comprises providing amicrofluidic network comprising a first region (e.g., region 1028) and amicrofluidic channel (e.g., microfluidic channel 1002) in fluidcommunication with the first region, flowing a first fluid (e.g., fluid1200) in a first direction in the microfluidic channel (e.g., in thedirection of arrow 1012), flowing a second fluid (e.g., fluid 1202) inthe first direction in the microfluidic channel, partitioning at least aportion of the first fluid at the first region so as to form a firstdroplet (e.g., droplet 1200-A) of the first fluid at the first region,and maintaining the droplet at the first region while fluid is flowingin the first direction. In some instances, forming and/or maintaining ofa droplet at a region is independent of flow rate of the first fluid inthe microfluidic channel.

The method described in connection with FIGS. 8 and 9 can allow storageof a fluid (e.g., a first fluid) at a region, followed by the creationof a droplet of the first fluid. This method contrasts with the methodsdescribed in connection with FIGS. 1-7, where droplets are first formedand then stored in the microfluidic network. In some embodimentsdescribed herein, a combination of both approaches can be used.

It should be understood that when droplet 1200-A is formed/positioned atregion 1028, the droplet may plug all or a portion of fluid path 1014and/or fluid restriction region 1024. For instance, in some cases, thedroplet plugs all of such fluid paths such that none of the fluidflowing in microfluidic channel 1002 passes through fluid restrictionregion 1024. In other embodiments, the droplet may plug only a portionof such fluid paths such that some fluid passes through fluidrestriction region 1024 even though the droplet is positioned at region1028. The amount of fluid flowing past the droplet may depend on factorssuch as the dimensions of fluid path portions 1014 and/or 1024, the sizeof the droplets, the flow rate, etc. As long as the droplet causes fluidpath 1014 to have a higher relative resistance to fluid flow than fluidpath 1018, a second fluid (e.g., fluid 1202) can bypass fluid path 1014and enter fluid path 1018.

As described above, fluid paths 1014 and 1018 may have differentresistances to fluid flow depending on whether or not a droplet has beenformed or positioned at region 1028. In the absence of a droplet atregion 1028, fluid path 1014 may be configured to have a lowerresistance to fluid flow than fluid path 1018. For example, greater than50%, greater than 60%, greater than 70%, greater than 80%, or greaterthan 90% of the fluid flowing in channel 1002 at upstream portion 1006may flow in fluid path 1014 compared to fluid path 1018. However, when adroplet has been formed, positioned and/or maintained at region 1028,fluid path 1014 may have a relatively higher hydrodynamic resistance.For example, less than 50%, less than 40%, less than 30%, less than 20%,or less than 10% of the fluid flowing in channel 1002 at upstreamportion 1006 may flow in fluid path 1014 compared to that of 1018. Insome cases, 100% of the fluid flowing in direction 1012 in microfluidicchannel 1002 flows in fluid path 1018 upon positioning of a droplet atregion 1028.

In some embodiments, partitioning of the first fluid into a firstportion at region 1028 to cause formation of droplet 1200-A of the firstfluid (e.g., a first droplet), can result in the droplet “recoiling”such that it has a slightly smaller volume than that of region 1028. Insome instances, this recoiling causes certain subsequent fluids flowingin the microfluidic channel (e.g., a third fluid that is miscible withthe first fluid) to not come into contact with the droplet stored atregion 1028. The third fluid may be in the form of, for example, a fluidstream or a droplet. If the third fluid is in the form of a seconddroplet, the second droplet may not come into contact with the firstdroplet. Accordingly, in certain embodiments, a third fluid (e.g.,second droplet or a fluid stream or portion of a fluid stream) does notphysically contact the first droplet after forming and/or positioning ofa first droplet in region 1028.

In other embodiments, a third fluid (e.g., second droplet or a fluidstream or portion of a fluid stream) comes into physical contact withthe first droplet as it bypasses the first droplet, however, due to suchminimal contact between the two fluids, the fluids do not coalesce.Accordingly, in some instances, the forming and/or positioning ofdroplets in the microfluidic network can take place without the use ofsurfactants. In other words, surfactants in either a fluid flowing inchannel 1002 and/or within the droplets is not required in order tostabilize the droplets and/or prevent the droplets or fluids fromcoalescing with one another during forming, positioning or carrying thedroplet in the microfluidic channel, and/or during maintaining thedroplets within a predetermined region within the microfluidic network.However, in instances where coalescence is desired (e.g., to allow areaction between reagents contained in two droplets), the microfluidicnetwork and methods for operating the network can be configured to allowsuch physical contact and/or coalescence between droplets.

In some embodiments, methods for positioning a droplet in a microfluidicnetwork include the steps of providing a microfluidic network comprisinga first region (e.g., region 1028 of FIG. 8A) and a microfluidic channelin fluid communication with the first region, flowing a first fluid(e.g., a fluid to be stored) in the microfluidic channel, partitioning afirst portion of the first fluid at the first region (at least in partthrough action of the second fluid) so as to form the first droplet atthe first region, and partitioning a second portion of the first fluidat the second region (at least in part through action of the secondfluid) so as to form the second droplet at the second region. In somecases, the first and second fluids are immiscible. The first droplet maybe formed, positioned and/or maintained at the first region while thefirst fluid and/or second fluid is flowing in the microfluidic channel.In some embodiments, forming, positioning and/or maintaining the firstdroplet at the first region does not require the use of a surfactant inthe first or second fluids.

Another method of the invention comprises providing a microfluidicnetwork comprising at least a first inlet to a microfluidic channel(e.g., positioned upstream of portion 1006 of FIG. 8), a first region(e.g., region 1028-A) and a second region (e.g., region 1028-B) forforming a first and a second droplet, respectively, the first and secondregions in fluid communication with the microfluidic channel. The methodcan include flowing a first fluid in the microfluidic channel andpartitioning a first portion of the first fluid at the first region, atleast in part through action of the second fluid, so as to form thefirst droplet at the first region. The method can also includepartitioning a second portion of the first fluid at the second region,at least in part through action of the second fluid, so as to form thesecond droplet at the second region. In some instances, the first and/orsecond droplets are maintained at their respective regions while fluidcontinues to flow in the microfluidic channel.

In some embodiments, a chemical and/or biological process and/or amanipulation process can be carried out in a droplet that is positionedat a region of a microfluidic network (e.g., droplet 1200-A of FIG. 8while the droplet is positioned in region 1028). For example, a fluidsample in the droplet may undergo a process such as diffusion,evaporation, dilution, and/or precipitation. The droplet may bemanipulated, for example, by changing the concentration of the fluidflowing in channel 1002 after the droplet has been formed/positioned atregion 1028. In other embodiments, region 1028 is in fluid communicationwith another fluidic channel, flow path, reservoir, or other structure,e.g., via a semi permeable membrane that may be positioned adjacent theregion (e.g., underneath or above region 1028), and manipulation of thedroplet can occur via such passages.

Microfluidic network 1000 of FIG. 8 may include additional regions 1001for forming/storing droplets. Accordingly, in some embodiments, a thirdfluid can be flowed in the microfluidic channel after flowing of thesecond fluid. The second and third fluids may be immiscible (while thefirst and third fluids may be miscible). The third fluid may bepartitioned at a second region (e.g., at least in part by flowing of afourth fluid past the second region) so as to form a second droplet atthe second region. Several droplets, each containing different fluidcompositions, can be formed and/or stored in regions of a microfluidicnetwork using this process.

In some embodiments, a fluid of defined volume (e.g., a “slug”) can beintroduced into microfluidic networks described herein and portions ofthe fluid can be partitioned into regions of the fluidic network. Forexample, a slug having a volume of X may be flowed into a network havinga plurality of sections 1001 (and regions 1028, having a volume of Y,where Y is less than X) and the slug can be partitioned at each region1028. The first region may be filled with the fluid in the amount ofvolume approximately Y (and forming a droplet of the fluid having avolume of approximately Y at the first region), while the remainder ofthe slug (X−Y or X minus Y) continues to flow down the network. A secondregion can then be filled with the fluid in the amount of approximatelyY (and forming a droplet of the fluid having a volume of approximately Yat the second region), while the remainder of the slug (X−2Y) continuesto flow down the network. This process can continue until the slug has avolume of zero. This method allows all of the fluid of the slug, e.g., asample fluid, to be used in the microfluidic network with no or minimalwaste.

As shown in FIG. 9, a microfluidic device 1210 may have a plurality ofregions 1001-A-1001-E for forming, storing, and/or maintaining droplets.Microfluidic channel 1012 may first be filled by flowing a filling fluid1011 in the direction of arrow 1012. Channel 1012 may then be filled byflowing a first fluid 1200 (e.g., in the form of a “slug”) in the samedirection. To form droplets of the first fluid, a second fluid 1202 maybe flowed in the same direction. At section 1001-A, second fluidbypasses fluid path 1014-A and enters fluid path 1018-A due to thepresence of the first fluid at region 1028-A. This causes thepartitioning of the first fluid and the formation of droplet 1220-A. Thesecond fluid continues to flow down the network, where it reachessection 1001-B. Due to the presence of first fluid at region 1028-B, thesecond fluid bypasses fluid path 1014-B and enters fluid path 1018-B.This process can continue until droplets are formed within regions ofsections 1001-C, -D, and -E.

In another aspect of the invention, microfluidic structures and methodsdescribed herein are designed for containing and positioning components(e.g., beads, cells, and other reactive or non-reactive components) inan arrangement such that the components can be grown, multiplied and/ormanipulated and then tracked even after one or more of these processeshas taken place.

As shown in the embodiment illustrated in FIG. 10, a microfluidicnetwork 1000 as described above may be used for positioning such andother components. FIG. 10A shows an empty microfluidic channel witharrows that show possible paths for fluid flow. As shown in FIG. 10B, afirst fluid 1200 containing reactive components 1230 and 1231 (e.g., abead comprising an antigen) is flowed into channel 1002 in the directionof arrow 1012. At upstream portion 1006, a first portion of the fluidflows into fluid path 1014, carrying with it reactive component 1230,while a second portion of the fluid flows into fluid path 1018, carryingwith it reactive component 1231. (A portion of fluid may flow into fluidpath 1014 before fluid path 1018 if fluid path 1014 is designed to havea lower hydrodynamic resistance than fluid path 1018.) Reactivecomponent may reside in region 1028 if, for example, the size of thereactive component does not allow it to pass through fluid restrictionregion 1024. In some embodiments, the fluid can continue to pass throughthis region even though reactive component is present in region 1028,although in other embodiments, there is little or no fluid flow throughthis region while the reactive component is present in this region.Accordingly, reactive component 1230 is positioned within region 1028 ofthe microfluidic network. In some embodiments, reactive component 1230can be maintained at the region even though fluid continues to flow inthe microfluidic network (e.g., in the direction of arrow 1012).

Accordingly, a method of the invention comprises providing amicrofluidic network comprising at least a first inlet to a microfluidicchannel, a first and a second region for positioning a first and asecond reactive component, respectively, the first and second regions influid communication with the microfluidic channel, wherein the firstregion is closer in distance to the first inlet than the second region,and flowing a first fluid comprising first and second reactivecomponents in the microfluidic channel. The method may also includepositioning the first reactive component at the first region,positioning the second reactive component at the second region, andmaintaining the first and second reactive components in the first andsecond regions, respectively, while a fluid is flowing in themicrofluidic channel.

As shown in the embodiment illustrated in FIG. 10C, reactive component1231 has now flowed past section 1001, while reactive component 1230remains at region 1028. A second fluid 1250 may be flowed into channel1002 in the direction of arrow 1012, e.g., to introduce another reactivecomponent into the microfluidic network. The second fluid may enter bothfluid path 1014 (including region 1028) and fluid path 1018. This secondfluid may be miscible with the first fluid and may contain a reactivespecies 1234 (e.g., a human-antibody). In some cases, reactive species1234 interacts (e.g., reacts, associates, or binds) with reactivecomponent 1230, e.g., as shown in FIG. 10D. In one particularembodiment, reactive component 1230 includes a binding partner that iscomplementary to that of reactive species 1234 (e.g., anantibody-antigen binding pair).

Next, a third fluid 1260 may be flowed in channel 1002 in the directionof arrow 1012, e.g., to introduce yet another reactive component intothe microfluidic network. The third fluid may enter both fluid path 1014(including region 1028) and fluid path 1018. This third fluid may bemiscible with the first and/or second fluids and may contain a reactivespecies 1236 (e.g., an enzyme-linked anti-human antibody). In somecases, reactive species 1236 interacts (e.g., reacts, associates, orbinds) with either reactive component 1230 or with reactive species1234, e.g., as shown in FIG. 10E. In one particular embodiment, reactivecomponent 1230 and/or reactive species 1234 includes a binding partnerthat is complementary to that of reactive species 1236 (e.g., anlabeled-antibody-antigen binding pair). Optionally, a fluid such as abuffer may be flowed in the microfluidic system as a washing step toreduce any non-specific binding.

Optionally, a fourth fluid 1270 (e.g., an oil or a gel) immiscible withthe third fluid may be flowed into channel 1002 in the direction ofarrow 1012. The fourth fluid may enter region 1028 and, using themethods described above in connection with FIGS. 8 and 9, a droplet(e.g., containing the reactive component(s)) may be formed at theregion, as shown in FIG. 10F.

In some embodiments, the positioning or presence of a reactive componentat region 1028 causes at least a portion of fluid path 1014 to beplugged such it has a higher hydrodynamic resistance, resulting in lessfluid flowing past fluid restriction region 1024 (relative to theabsence of the component). This may cause fluid path 1014 to have ahigher hydrodynamic resistance than fluid path 1018. As a result, whensubsequent fluids (including additional reactive components) are flowedin the direction of arrow 1012, at least a portion of the subsequentfluids (and reactive components) may bypass flow path 1014 and enterflow path 1018, which now has a lower hydrodynamic resistance than fluidpath 1014. Accordingly, once region 1028 has been “filled” with areactive component, other regions downstream can be filled with reactivecomponents. This allows a plurality of reactive component to begenerated/stored sequentially down the length of the channel.

Reactive components may interact with reactive species by variousmethods. For example, interaction may be between a corresponding pair ofcomponents that exhibit mutual affinity or binding capacity, typicallyspecific or non-specific binding or interaction, including biochemical,physiological, and/or pharmaceutical interactions. Biological bindingdefines a type of interaction that occurs between pairs of componentsincluding proteins, (e.g., adhesion proteins), nucleic acids,glycoproteins, carbohydrates; hormones and the like. Specific examplesinclude cell (e.g., cell surface receptor)/protein, adhesionprotein/integrin, antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc.

By carrying out the methods described above in connection with FIG. 10in a device including a plurality of sections 1001, a plurality ofreactive components can be positioned, stored, and/or maintained in thedevice. This can allow performance of a variety of reactions or analysessuch as assays (e.g., ELISA) and PCR.

In some embodiments, reactive components such as cells can beencapsulated in a droplet (e.g., as part of a water-in-oil emulsions).The droplet may contain one or more reactants, nutrients, or drugs. Thedroplets may act as, for example, picoliter-scale reaction microvessels.Encapsulation can enable reactions involving, for instance, singlemolecules, such as in vitro translation of proteins from single genes,or DNA amplification of single genes by polymerase chain reaction (PCR).Droplets also provide an excellent method for studies of single,isolated cells, compounds they secrete, and/or growth rates of singlecells.

A method involving the use of droplets as microvessels may requireincubation and imaging of droplets. Certain existing methods allowcollection and imaging of droplets, however, it is difficult to maintainthe identity of the droplets. Using the articles and methods describedherein, these challenges can be overcome in certain embodiments sinceeach droplet may be associated with a particular region of themicrofluidic device.

In one particular embodiment, cells can be encapsulated in a gel byusing methods and articles described herein: By encapsulating cells in ahydrogel functionalized with the required reactants, heterogeneousassays can be performed in a three-dimensional scaffold. Placinghydrogels in a microfluidic network such as the one shown in FIG. 10 canallow reactions in a population of individual gels to be tracked overtime.

In some embodiments, the microfluidic systems and methods describedabove and below can be used for containing and positioning cells, whichcan be grown, multiplied and/or manipulated in the microfluidic system.The cells (and their progeny) can be tracked even after one or more ofthese processes has taken place.

Genetically identical cells show phenotypic variation, for exampleduring differentiation, development, and in response to environmentalstress. The mechanisms of phenotypic variation are not fully understood,yet they are central to our understanding of major questions in biologysuch as differentiation and ageing. Gene expression patterns can bepassed on to progeny cells, however, the mechanisms of non-geneticinheritance are not fully understood. To study phenotypic switching andepigenetic mechanisms of gene regulation, the systems and methodsdescribed herein can be used to study lineages of single cells and theirprogeny. For example, in some embodiments, single yeast cells can betrapped in long, thin chambers of a microfluidic device and the cellscan be cultured so they grow in a line. This configuration facilitatesthe following of lineages deriving from a single progenitor cell. Cellscan grow and divide in the chambers with typical doubling times, e.g.,under constant flow conditions. As described in more detail below,generations of yeast cells can be tracked. The frequency of phenotypicswitching of, for example, the GFP (green fluorescent protein)-fusionprotein and PHO84-GFP, a high affinity phosphate transporter, can alsobe studied using the methods and devices described herein.

The methods can be applied to yeast cells, and more broadly to anysuspension cell including blood cells, stem cells, and other mammaliancells that grow in suspension. This methods are also useful for stainingsingle cells and their lineages: microcolonies deriving from singlecells are cultured in the chambers, and then stained. Quantitativeimaging can be applied to study correlations between stained entities(protein amount or localization, chromosome position, etc) and singlecell genealogy or replicative age. Moreover, the antibodies and DNAprobes required for immunofluorescence and fluorescence in situhybridization (FISH) are expensive; performing these assays inmicrofluidic devices described herein reduces the volume and thus costof the required reagents. The methods and devices described herein canalso enable imaging suspension cells over time.

FIG. 11 is an example of a microfluidic system that can be used forcontrollably positioning cells or other components in certain regions ofthe system. As shown in the embodiment illustrated in FIG. 11,microfluidic system 1300 includes a plurality of chamber units 1308(and/or, in other embodiments, chamber units 1309) positioned inparallel. The chamber units include a chamber inlet 1312, a chamberoutlet 1324, and a chamber 1320 (having a length 1321) in fluidcommunication with the chamber inlet and the chamber outlet. As shown inthe exemplary embodiment of FIG. 11, chamber 1320 is in the form of along linear channel that can be used to position one or more components;however, it should be understood that any suitable shape and/orconfiguration of the chamber can be used in embodiments describedherein. For instance, a chamber channel may be serpentine, curved,tapered, in the form of a reservoir, and the like.

At a downstream portion of chamber 1320 is positioned a fluidrestriction region 1328, which, in some embodiments, may be in the formof a narrow channel. This fluid restriction region may allow certaincomponents and/or fluids to pass therethrough, while inhibiting othercomponents and/or fluids from passing therethrough, such that certaincomponents and/or fluids are retained in chamber 1320. In some cases,once one or more components are positioned in a chamber, the position ofthe component(s) can be maintained in the chamber even during subsequentfluid flow in the chamber. This may occur, in some cases, if thecomponent has an average cross-sectional area that is larger than across-sectional area of fluid restriction region 1328.

A fluid restriction region may be positioned at an upstream portion, adownstream portion, or in between an upstream and a downstream portionof the chamber. In some cases, a fluid restriction region is positionedbetween a chamber inlet and a chamber outlet. In other cases, a fluidrestriction region is positioned outside of the chamber. In oneembodiment, a fluid restriction region is positioned immediatelyadjacent a chamber outlet.

It should be understood that any suitable structure can be used as fluidrestriction region 1328, which may have a higher hydrodynamic resistanceand/or a smaller cross-sectional area for fluid flow than a regionimmediately upstream or downstream of the fluid restriction region. Forinstance, fluid restriction region 1328 may be in the form of a narrowfluid path or a channel having the same dimensions as chamber 1320, buthaving an obstruction (e.g., posts or a valve) positioned in or at thatregion. In other embodiments, fluid restriction region 1328 may comprisea porous membrane, a semi-permeable plug (e.g., a gel), a valve, oranother structure. In some cases, a fluid restriction region is a narrowchannel portion that does not include a gel or other structure disposedtherein; e.g., the size of the opening of the fluid restriction regionalone can be used to trap, immobilize, and/or position components in achamber fluidly connected to the fluid restriction region.

As shown in the illustrative embodiment of FIG. 11, chamber unit 1308includes a chamber unit outlet 1344, which is the same as the outlet offluid restriction region 1328. In chamber 1309, however, chamber unitoutlet 1316 is downstream of fluid restriction region 1328. Otherconfigurations of the chamber unit outlet are also possible.

Also included in chambers 1308 and 1309 are chamber bypass channels1330, which extend from a portion of chamber 1320. One or more chamberbypass channels may extend from an upstream and/or downstream portion ofthe chamber. In some cases, a chamber bypass channel extends from thechamber between a chamber inlet and a chamber outlet (e.g., asillustrated in FIG. 11). In other cases, an inlet of the chamber bypasschannel intersects an inlet of the chamber at an intersection.

In some embodiments, a chamber bypass channel has a lower hydrodynamicresistance than a chamber (e.g., prior to and/or after a component hasbeen positioned in the chamber). As described in more detail below, thiscan allow more fluids and/or components to flow into the chamber bypasschannel than the chamber. This arrangement may be useful forapplications where it is desirable to position only one, or perhaps afew, components in the chamber. In certain embodiments where it isdesirable to flow fluids and/or components out of the chamber bypasschannel, the chamber bypass channel does not include any fluidrestriction regions. This configuration may prevent any fluids and/orcomponents from being immobilized in the chamber bypass channel. Inother embodiments, however, a chamber bypass channel may include one ormore fluid restriction regions.

As shown in the exemplary embodiment of FIG. 11, a plurality of chamberinlets 1312 may be fluidly connected to one or more feed channels 1340,which can allow delivery of fluid and/or components to the chamberinlets. Similarly, chamber outlets 1316 may be connected to one or moredrain channels 1344. This arrangement can allow fluids and/or componentswhich are not positioned in chamber 1320 to be collected and,optionally, recycled back into the microfluidic system (e.g., by fluidlyconnecting a drain channel to a device inlet). Although FIG. 11 showseach of the chamber inlets and chamber outlets being connected to feedchannel 1340 and drain channel 1344, it should be understood that otherconfigurations are possible. For example, in some embodiments, a firstset of chamber units is connected to a first feed channel, and a secondset of chamber units is connected to a second feed channel. In otherembodiments, chambers or chamber units may be connected in series suchthat a chamber unit outlet of a first chamber is connected to a chamberinlet of a second chamber. In yet other embodiments, an outlet of achamber bypass channel of a first chamber can be connected to an inletof a second chamber. For example, chamber bypass channel 1330 of chamberunit 1308 may include a chamber bypass channel outlet 1332 that isconnected to a chamber inlet 1312 of a second chamber, or between achamber inlet and a chamber outlet of a second chamber, instead of beingconnected to drain channel 1344. In yet another embodiment, an outlet ofa chamber bypass channel can be connected to a feed channel. In somecases, a combination of the configurations described above can beincluded in a single microfluidic system. Accordingly, differentcombinations of chambers positioned in series, parallel, and/or otherarrangements can be included in microfluidic systems described herein.

A microfluidic device may include, for example, at least 10, at least25, at least 50, at least 75, at least 100, at least 150, at least 200,at least 500, or at least 1,000 chambers or chamber units, which may bepositioned in series and/or in parallel. The number of chambers orchamber units, in some cases, may depend on and/or may be limited by thesize of the device, the size of the chamber units, and/or theapplication. Optionally, one or more chambers or chamber units may belabeled, e.g., using labels 1372, to identify the chambers or chamberunits. Accordingly, any suitable number of chambers or chamber units maybe included in a device. In addition, a device may include one or moremicrofluidic systems 1300 which can allow, for example, experimentsunder conditions or parameters to be performed simultaneously.

Microfluidic system 1300 may also include one or more device inlets(inlets 1346 and 1348) for introducing one or more fluids into themicrofluidic system. As shown in FIG. 11, a filter 1350 may optionallybe positioned downstream of inlet 1348 (and/or inlet 1346) and upstreamof the chambers and feed channel 1340. This filter can allow, forexample, components of certain sizes to be flowed into the chambers,while restricting components of other sizes from entering into thechambers. For example, the filter may allow single components having aparticular size to be flowed into the chambers, while restrictingagglomeration of components, which have a larger size, from enteringinto the chambers.

Inlet 1346 may be connected to two channel portions 1362 and 1364, andinlet 1348 may be connected to channel portion 1360. As illustrated,channel portions 1360, 1362, and 1364 may be arranged in a flow focusingarrangement. This arrangement can facilitate focusing of a componentwithin channel portion 1360 towards a center portion of a fluid streamduring laminar flow, e.g., when the channel portions intersect atintersection 1366 to form laminar streams of fluid. The fluid can thenbe introduced into microfluidic channel 1340 via inlet 1368. Otherconfigurations of inlets are also possible.

Fluids flowing in channel portions 1362 and 1364 may be immiscible witha fluid flowing in channel portion 1360, which may allow formation ofdroplets of the fluid flowing in channel portion 1360 at or near inlet1368 in some embodiments. In other embodiments, the fluids in channelportions 1360, 1362 and 1364 are miscible.

As described herein, a microfluidic system may be used to position oneor more components in one or a series of regions (e.g., “microwells” or“chambers”). A variety of different components can be positioned at aregion, including, for example, droplets, cells (e.g., bacterial, yeast,mammalian and stem cells), beads, tissues, and other entities. Thenumber of components positioned at a region may depend on the size ofthe region. In some cases, a region (or a component) may have adimension, such as a length, width, and/or height, of less than or equalto about 500 μm, less than or equal to about 250 μm, less than or equalto about 100 μm, less than or equal to about 50 μm, less than or equalto about 25 μm, less than or equal to about 10 μm, less than or equal toabout 8 μm, or less than or equal to about 1 μm. The volume of theregion (or component) can also vary; for example, the region (orcomponent) may have a volume of less than or equal to about 50 μL, lessthan or equal to about 10 μL, less than or equal to about 1 μl, lessthan or equal to about 100 nL, less than or equal to about 10 nL, lessthan or equal to about 1 nL, less than or equal to about 100 μL, or lessthan or equal to about 10 μL. In certain embodiments, a region (or acomponent) may have a length of at least 1 μm, at least 10 μm, at least25 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 250μm, at least 500 μm, or at least 1,000 μm. Long regions may be suitablefor positioning a large number of components at the region.

To position components in chamber of microfluidic system 1300, a firstfluid stream including a plurality of components (e.g., cells) may beintroduced into inlet 1348. Agglomeration of cells may be captured byfilter 1350, e.g., a size exclusion filter, while allowingun-agglomerated cells to pass therethrough. A second fluid (e.g., abuffer solution) may be introduced into inlet 1346, and this fluid canflow into channel portions 1362 and 1364. The first fluid including thecomponents may be focused as they pass through inlet 1368 into feedchannel 1340. Since feed channel 1340 fluidly connects to a series ofchamber inlets, the feed channel can distribute the fluids and thecomponents to various chambers.

The geometry of the channels of the microfluidic system, the flow rateof the first and second fluids, and the concentration (or density) ofthe components in the first fluid can be chosen so as to allow deliveryof a certain number of components into each of the chambers. Forexample, these parameters can be chosen such that at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of the chambers containa predetermined number of components positioned therein. For instance,in one embodiment, only a single component is delivered to at least 50%,at least 60%, at least 70%, at least 80%, or at least 90% of thechambers of a microfluidic system. This can be achieved, in part, bydesigning the chambers to have a cross-sectional area on the order ofthe cross-sectional area of the component to be positioned. In addition,fluid restriction region 1328 may be designed such that introduction ofa single component into the chamber channel substantially blocks orreduces fluid flow through this region. (In other embodiments, the fluidrestriction region may be designed such that the introduction of exactlytwo components, or exactly three components, etc., causes substantialblockage of fluid flow through the fluid restriction region.) Thisblockage or reduction in fluid flow can result in the chamber being“filled”, causing the chamber to have a higher hydrodynamic resistancethan an empty chamber. Thus, fluid and components flowing in channel1340 may prefer to flow into empty chambers (and/or through a chamberbypass channel) having a lower hydrodynamic resistance. The flow rateand the concentration (or density) of components in the fluids can alsobe adjusted to control the number of components in each chamber. Thiscontrol can allow, for example, multiple experiments to be performed inparallel under similar or the exact same conditions.

Accordingly, a method may include flowing a fluid containing a pluralityof components in a microfluidic system comprising a chamber having aflow direction, a chamber inlet, a chamber outlet, and a chamber bypasschannel extending from the chamber between the chamber inlet and thechamber outlet. The method may also include positioning a component inthe chamber, the chamber having a cross-sectional area, perpendicular tothe flow direction, less than 2 times the largest cross-sectional areaof the component perpendicular to the flow direction. A fluid may beflowed through the chamber while maintaining the component at itsposition in the chamber, and a portion of the plurality of componentsmay be flowed in the chamber bypass channel.

As described herein, the dimensions of the chamber and fluid restrictionregion 1328 may be chosen such that a component, or a series ofcomponents, can be positioned, maintained, or trapped in the chamberchannel without exiting the chamber via fluid restriction region 1328.In some cases, this positioning, maintaining, or trapping take placeeven while fluid continues to flow through fluid restriction region1328. The fluid may be flowed at a substantially constant flow rate, atvarying flow rates, or flow may take place periodically. This fluid flowmay be important for applications that involve treating or manipulatingthe component(s) after it has been positioned in the chamber channel.For example, a continuous or periodic flow of nutrients may be suppliedto cells that are trapped in a chamber channel. These nutrients canfacilitate the growth and/or multiplication of the cells. Flow of dyescan allow the cells to be tagged and identified. In other embodiments,components that are positioned in a chamber can be subjected to a stress(e.g., different buffers, nutrients, or exposure to drugs) and theresponse of different components to the stress can be determined. Otherapplications are described in more detail below.

Flow of fluid (e.g., media) through the chambers is important for cellculture and also allows the cells to be exposed to controlledenvironmental changes, as well as immunostaining. Immunofluorescence ofsuspension cells is generally challenging. For yeast, the cell wall istypically enzymatically removed, and the cells are attached to asubstrate using a chemical crosslinker. The cells are then fixed andpermeabilized. Other suspension cells are typically centrifuged onto asurface-treated, glass microscope slide before staining. Moreover,immunostaining techniques for suspension cells are typically notcompatible with live cell imaging. Using the methods and systemsdescribed herein, however, live cells can be positioned and trackedcontrollably in regions of a microfluidic device, and environmentalconditions can be varied, for example, by changing the fluids flowed inthe region.

Furthermore, in some embodiments, the dimensions of the chamber can bechosen such that components in the chamber are positioned in apredetermined configuration. For example, the dimensions of the chambermay be chosen such that a series of components are generally aligned inthe chamber. As used herein, “generally aligned” means no greater than20% of the components in a chamber have the same position along thedirection of bulk fluid flow in the chamber. The position of thecomponents along the direction of bulk fluid flow can be determined bytaking the cross-section of the component, perpendicular to thedirection of (bulk) fluid flow in the chamber, which cross-sectionpasses through the center of the component between two opposed points ofa surface/surfaces of the component. For example, as shown in theillustrative embodiment of FIG. 12A, chamber 1320 includes a pluralityof components 1390 that are generally aligned because less than 20% ofthe components in the chamber have the same position along the directionof bulk fluid flow, indicated by arrow 1395. The cross-section 1392-A ofcomponent 1390-A, perpendicular to bulk fluid flow, is shown to passthrough the center of the component, and the component has a position1391 (relative to the direction of bulk fluid flow). Component 1390-Ahas a different position along the direction of bulk fluid flow thanthat of components 1390-B or 1390-C. Component 1390-B has across-section 1392-B perpendicular to bulk fluid flow and component1390-C has a cross-section 1392-C perpendicular to bulk fluid flow. Bothof these cross-sections, and therefore both components 1390-B and1390-C, have the same position relative to the direction of bulk fluidflow.

In some cases, no greater than 15%, 10%, 5%, or 2% of the components ina chamber have the same position along the direction of bulk fluid flowin the chamber. For example, sometimes 100% of the components in achamber have different positions along the direction of bulk fluid flowin the chamber. When no greater than 5% of the components in a chamberhave the same position along the direction of bulk fluid flow in thechamber, the components may be aligned “in a single line” in thechamber, as shown in the embodiment illustrated in FIG. 12B.

Aligned or generally aligned components can assist in identification ofthe components in a chamber. For example, if the components are cellsderived from one cell, this configuration may be useful for tracking thelineology of the cells. In order to achieve the positioning of alignedor generally aligned components, the chamber may have a cross-sectionalarea perpendicular to the (bulk) flow direction that is, for example,less than 2 times the largest cross-sectional area of the componentperpendicular to the (bulk) flow direction. In some cases, the chamberhas a cross-sectional area, perpendicular to the (bulk) flow direction,that is less than 10 times, 7 times, 5 times, or 3 times thecross-sectional area of a component perpendicular to the (bulk) flowdirection.

In some embodiments, at least 50%, at least 60%, at least 70%, at least80%, or at least 90% of the components in a chamber have a largestcross-sectional area, perpendicular to the flow direction, of between0.1 and 1.0 times (or between 0.3 and 1.0 times, or between 0.5 and 1.0times) the cross-sectional area of the chamber perpendicular to the flowdirection. It should be understood that the cross-sectional area may bestatic or dynamic, e.g., depending on the materials used to form thechamber. For example, a chamber may comprise an elastomer that allows itto expand upon having a high pressure in the chamber, or a largecomponent positioned in the chamber. If a component completely blocks aportion of the chamber, even causing the chamber to expand, the largestcross-sectional area of the component perpendicular to the flowdirection may be the same as (e.g., 1.0 times) the cross-sectional areaof the chamber perpendicular to the flow direction.

In one particular embodiment, a system comprises a microfluidic devicecomprising an inlet, an outlet, a chamber having a (bulk) flowdirection, and a flow restriction region fluidly connected to the outletof the chamber. A plurality of cells may be generally aligned in thechamber, wherein at least 80% of the cells have a largestcross-sectional area, perpendicular to the flow direction, of between0.1 and 1.0 times the cross-sectional area of the chamber perpendicularto the flow direction. In other embodiments, these dimensions arebetween 0.3 and 1.0 times, or between 0.5 and 1.0 times thecross-sectional area of the chamber perpendicular to the flow direction.The flow restriction region may be constructed and arranged to allow afluid but not the cells to pass therethrough, thereby allowing the cellsto be maintained in their positions in the chamber.

In certain embodiments, the chamber has a cross-sectional area having adifferent shape than a cross-sectional area of the component. Forinstance, the component may be round and the cross-sectional area of thechamber channel may be square. This shape difference can allow thecomponents to be trapped in the chamber, but may allow fluids to passthe component at the corners of the chamber. Thus, a component may havea largest cross-sectional area, perpendicular to the flow direction, ofless than 1.0 times, less than 0.8 times, or less than 0.5 times thecross-sectional area of the chamber perpendicular to the flow direction.

A cross-sectional area (or an average cross-sectional area) of a chamber(or a component) perpendicular to the (bulk) flow direction may be, forexample, about 10,000 μm² or less, about 5,000 μm² or less, about 2,500μm² or less, about 1,000 μm² or less, about 500 μm² or less, about 250μm² or less, about 100 μm² or less, about 50 μm² or less, about 30 μm²or less, about 20 μm² or less, about 10 μm² or less, or about 5 μm² orless. As mentioned above, the cross-sectional area of a chamber may bechosen depending on factors such as the size of the components at or tobe flowed in the chamber. The ratio of average cross-sectional areas ofthe chamber and the components (both measured perpendicular to the(bulk) flow direction) may be, for example, less than 20:1, less than10:1, less than 5:1, less than 3:1, less than 2:1, or less than 1.5:1.Additionally or alternatively, the cross-sectional area of a chamber maybe chosen depending on factors such as the cross-sectional area of afluid restriction region, the number and type of components, and/or thefluids to be flowed in the system. The ratio of average cross-sectionalareas of the chamber and a fluid restriction region (both measuredperpendicular to the (bulk) flow direction) may be, for example, greaterthan 1:1, greater than 2:1, greater than 5:1, greater than 10:1, orgreater than 25:1. In some cases, the ratio is between 3:1 and 8:1.

A cross-sectional area (or an average cross-sectional area) of a fluidrestriction region (or the average cross-sectional area of the combinedfluid paths in the case of a porous membrane or other structure having aplurality of fluid paths) may be, for example, about 5,000 μm² or less,about 2,500 μm² or less, about 1,000 μm² or less, about 500 μm² or less,about 250 μm² or less, about 100 μm² or less, about 50 μm² or less,about 30 μm² or less, about 20 μm² or less, about 10 μm² or less, orabout 5 μm² or less, or about 1 μm² or less. In some cases, thecross-sectional area of a fluid restriction region may be chosendepending on factors such as the cross-sectional area of the chamber,the number, type and size of components, and the fluids to be flowed inthe system.

In some embodiments, cells are positioned in a chamber and are allowedto multiply. Further analyses may involve comparing a characteristic ofa first cell from a first generation to a characteristic of a secondcell from a second generation. Additionally or alternatively, the cellscan be subjected to a stress and/or a condition, and the response of oneor more cells to the stress or condition can be determined. For example,comparative analyses may be performed by determining the response of atleast two cells to a stress under controlled conditions.

Understanding how populations of single cells respond to environmentalchanges provides critical insights into variability in biologicalresponse, from differentiation to multi-drug resistance. Imagingpopulations of single cells is challenging for cells that are notadherent. Suspension cells can be trapped in wells, however, it may bedifficult to image non-adherent cells under changing environmentalconditions. Budding yeast cells can adhere to treated surfaces (e.g.concanavalin A or agar pads), however, newly budded yeast cells may notadhere under flow conditions. Moreover, yeast cells bud in multipledimensions (e.g., different focal planes), challenging the analysis ofpopulations of cells over time. Using the devices described herein,individual cells can be placed in an array, cultured, imaged in a singlefocal plane, and can be subjected to changing conditions. Furthermore,the microfluidic systems described herein can be fabricated on amicroscope slide, which can facilitate imaging and viewing of thesystems. Accordingly, multiple flow experiments can be performed inparallel for the simultaneous study and/or comparison of different flowconditions or cell types.

It should be understood that a variety of different cells can be used indevices described herein. Non-limiting examples include yeast cells,bacteria, stem cells, and suspension cells. In some embodiments, thecells are round while the chambers are square, allowing for flow pastthe cells. Other geometries of chambers can also be used.

Certain existing methods for time-lapse microscopy of budding yeastunder flow require that the cells are wedged into a chamber much smallerthan the height of the cells themselves, so that they do not move duringflow; however in such chambers, the cells are subjected to mechanicalstress, which may affect the observed response. In addition, certainother existing methods allow cells to grow in a 2D plane, but the cellsare randomly distributed in chambers and cannot be subjected tocontrolled flow conditions, making it difficult to obtain data on manysingle cell lineages in a single experiment as microcoloniesinterdigitate as they grow. Using the devices described herein can allowfor fixing and staining of populations of single cells without imposingmechanical stress on the cells, while allowing precise positioning andcontrol of the numbers of cells, and manipulation of cells, in chambers.

FIGS. 13A and 13B are bright field and fluorescence images,respectively, showing a plurality of chambers units 1308 includingchambers 1320. An array of single, fluorescently tagged cells 1398 ispositioned in the chambers. A group of cells 1399 is present in one ofthe chambers, and some chambers do not include any cells. The cells areprevented from reaching chamber outlet 1316 or from flowing downstreamby fluid restriction region 1328.

FIGS. 14A and 14B are bright field and fluorescence images,respectively, showing a plurality of cells in chambers 1308, which cellshave grown from single cells similar to the ones shown in FIGS. 13A and13B. Thus, cells can be multiplied in a chamber to form at least 2, atleast 5, at least 10, at least 20, at least 50, or at least 100 progenyfrom a single cell. As shown in FIGS. 14A and 14B, the cells aregenerally aligned as they multiply.

As shown in the embodiments illustrated in FIGS. 15A-15B, cells 1498 canbe positioned and/or grown in single lines in chambers 1410 of amicrofluidic network 1400. In some embodiments, microfluidic network1400 may have the same configuration as network 1000 of FIG. 10, andchambers 1410 may be equivalent to regions 1028. Fluid restrictionregion 1420 restricts cells 1498 from flowing downstream in thedirection of arrow 1430. A plurality of chambers 1410 may be positionedin series (as shown in FIGS. 15A-15B), and/or in parallel. In thisparticular embodiment, yeast cells (S. cerevisiae, s288c) are positionedin the form of a line in chamber 1410 at time=0 (FIG. 15A). The cellsare round while the channels have a cross-sectional area in the shape ofa square, so media (e.g., rich yeast media, YPD) can be continuouslyflowed through the channels (e.g., in the direction of channel 1430,although the reverse direction is also possible in certain embodiments).Since the channels are as wide as the cells, the cells are constrainedto grow in a line. FIGS. 15A-15B show a time-course of imagesillustrating cell growth.

As shown in the embodiment illustrated in FIGS. 16A-16C, generations ofcells 1498 cultured in a region of a microfluidic device can be tracked.FIG. 16A shows yeast cells expressing a fluorescent protein (S.cerevisiae, HXK1-GFP), which are grown in a chamber 1410. The design ofthis microfluidic network (e.g., microfluidic network 1000 of FIG. 10)provides an efficient method for making an array of single cells inregions of the device. These particular images were imaged by confocallaser scanning microscopy after ˜20 hour incubation and growth showsvariations in levels of gene expression (proportional to fluorescenceintensity) through generations of cells. Cells can also be stained byother fluorescent probes in these chambers.

FIGS. 17A-17C show additional photographs of a chamber of a microfluidicnetwork that can be used for growing and imaging cells. Here, thephotographs show yeast cells expressing a fluorescent protein (S.cerevisiae, HXK1-GFP) after growth in a region of a microfluidic device.These regions can allow for yeast cells, including newly budded yeastcells, to be imaged over time under flow. This enables analysis ofvariations in response of populations of single cells to changingenvironmental conditions.

FIGS. 18A-18C show the tracking of a lineage of yeast cells in achamber. As shown in FIG. 18A, at time=0, cells 1 and 1-1 are positionedin chamber 1450 (where cell 1-1 previously budded from cell 1). As shownin FIG. 18B, the cells have multiplied further. Because the cells canbud to the right or left of a cell, cell 1-1 is now shifted and ispositioned at a downstream portion of the chamber at time=480 min. Thetracking of the cells can be performed by time lapse microscopy and achart, such as the one shown in FIG. 18C, can be drawn based on thisdata.

As described herein, cells (or other components) can be labeled with anidentifier (e.g., a dye, a probe, etc.) that identifies a particularportion of the cell and/or a particular process occurring in the cell.For example, cells labeled with GFP may show a certain level of geneexpression within the cell. By combining this identification techniquewith methods described herein (e.g., the multiplication and tracking ofcells in a chamber), gene expression of a lineage of cells can betracked as a function of a cell's replicative age or history. Inaddition, these techniques may be useful for studying phenomena such asthe age effects of DNA repair, how cells of different age respond tostress (e.g., exposure to different growth conditions, to a drug, and/orto a change in temperature (e.g., a heat shock), the stress of being ina densely packed environment; etc.), how gene expression levels dependon the family history of cells, and frequency of cell phenotypeswitching events. In some cases, these and other phenomena can bestudied as a function of a cell's replicative age or history using thesystems and methods described herein.

FIG. 19 shows a plurality of cells in a chamber with differentintensities of fluorescence (the higher then number meaning greaterfluorescence); these different intensities show that each of the cellshas a different level of gene expression. The different fluorescentintensities of the cells can also aid in the tracking the cells (e.g.,to form the chart shown in FIG. 18C) since each cell is “labeled” byhaving a different intensity.

FIGS. 20A and 20B show yeast cells grown in a chamber 1452 having aplurality of branching channels 1452 in the form of a grid. Areas 1456do not contain channels and, therefore, these areas do not containcells. FIGS. 20A and 20B are inverted fluorescence images (e.g., cellsthat are dark are very bright with GFP). The cells are grown indifferent media: YPD (FIG. 20A) and synthetic dextrose media, SD (FIG.20B). In the YPD media, all cells are “ON” (e.g., they fluoresce)because they all express pPHO84-GFP (i.e., GFP expressed under thecontrol of the PHO84 promoter, where Pho84 is a high-affinity phosphatetransporter). In the SD media (FIG. P2), some cells are “ON” and someare “OFF”. These figures show that the type of media can cause cells tovary gene expression. These conditions can be varied and studied usingmicrofluidic systems described herein.

FIG. 21 shows cells grown in SD media and then cultured in the chambersof a microfluidic system similar to the one shown in FIG. 11. In theseconditions, some cells (cells 1460) are “ON” and others (cells 1462) are“OFF”. These lineages of cells can maintain the same phenotype for atleast 7 generations.

In some cases, certain strains of the GFP-fusion collection showed abimodal distribution of phenotypes (“ON” vs. “OFF”) after culture fromthe harsh climate of a freezer. Despite being genetically identical,these cells switch phenotype, and their progeny inherit this phenotype;this provides a model system to study epigenetic mechanisms in generegulation. The systems and methods described herein can be used toidentify colonies and/or conditions in which cells switch. Thisinformation may be relevant to the understanding of gene regulation inyeast cells, and more broadly, to the understanding of epigenetictimescales in adaptation and differentiation.

FIG. 22 shows a FISH staining using cDNA probes that bind to particularsequences of the cells' DNA. In this particular figure, the cDNA probeslabeled the telomeres of the cells. The staining process involvedheating the cells in the channel.

FIGS. 23A-23C show a screen of a GFP library of yeast cells positionedin a chamber. The cells were grown from a single cell that was under thestress of being grown in a densely packed environment. All cells werelabeled with GFP, however, the cells show different levels of geneexpression (and, therefore, have different fluorescent intensities) dueto a change in environmental conditions (e.g., exposure to a differentbuffer). FIG. 23A is a fluorescent image, FIG. 23B is a brightfieldimage, and FIG. 23C is an overlay of fluorescent and brightfield images.

FIGS. 24A-24E show different levels of gene expression in cells that areexposed to various buffers. Yeast cells were labeled with HXK1-GFP(hexokinase isoenzyme). FIGS. 24A-24C show cells exposed to a syntheticdextrose buffer. Different cells had different levels of gene expressionin this buffer. FIG. 24D shows cells exposed to YP-glycerol and FIG. 25Eshows cells in a YP-galactose buffer; all of the cells had high levelsof gene expression in these two buffers.

In other experiments, yeast cells were exposed to differentconcentrations of phosphate. High concentrations of phosphate caused thecells to turn “OFF”, e.g., the cells having low levels of geneexpression. Low concentrations of phosphate caused the cells to turn“ON”, e.g., the cells having high levels of gene expression.Intermediate concentrations of phosphate caused the cells to enter intoa bistable system where some progeny turned “ON”, and some turned “OFF”,the different levels switching every few generations.

In some embodiments, regions of a fluidic network such as microchannels,microwells, and/or chambers are defined by voids in the structure. Astructure can be fabricated of any material suitable for forming afluidic network. Non-limiting examples of materials include polymers(e.g., polystyrene, polycarbonate, PDMS), glass, and silicon. Those ofordinary skill in the art can readily select a suitable material basedupon e.g., its rigidity, its inertness to (i.e., freedom fromdegradation by) a fluid to be passed through it, its robustness at atemperature at which a particular device is to be used, itshydrophobicity/hydrophilicity, and/or its transparency/opacity to light(i.e., in the ultraviolet and visible regions).

In some instances, a device is comprised of a combination of two or morematerials, such as the ones listed above. For instance, the channels ofthe device may be formed in a first material (e.g., PDMS), and asubstrate can be formed in a second material (e.g., glass).

Fluid channels and/or components described herein may have maximumcross-sectional dimensions less than 2 mm, and in some cases, less than1 mm. In one set of embodiments, all fluid channels containingembodiments of the invention are microfluidic or have a largestcross-sectional dimension of no more than 2 mm or 1 mm. In anotherembodiment, the fluid channels may be formed in part by a singlecomponent (e.g., an etched substrate or molded unit). Of course, largerchannels, tubes, chambers, reservoirs, etc. can be used to store fluidsin bulk and to deliver fluids to components of the invention. In one setof embodiments, the maximum cross-sectional dimension of the channel(s)or regions described herein are less than about 500 microns, less thanabout 200 microns, less than about 100 microns, less than about 50microns, or less than about 25 microns. In some cases the dimensions ofthe channel may be chosen such that fluid is able to freely flow throughthe article or substrate. The dimensions of the channel may also bechosen, for example, to allow a certain volumetric or linear flowrate offluid in the channel. Of course, the number of channels and the shape ofthe channels can be varied by any method known to those of ordinaryskill in the art. In some cases, more than one channel or capillary maybe used. For example, two or more channels may be used, where they arepositioned inside each other, positioned adjacent to each other,positioned to intersect with each other, etc.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channels of the device may be hydrophilic or hydrophobic in order tominimize the surface free energy at the interface between a materialthat flows within the channel and the walls of the channel. Forinstance, if the formation of aqueous droplets in an oil is desired, thewalls of the channel can be made hydrophobic. If the formation of oildroplets in an aqueous fluid is desired, the walls of the channels canbe made hydrophilic.

In some cases, the device is fabricated using rapid prototyping and softlithography. For example, a high resolution laser printer may be used togenerate a mask from a CAD file that represents the channels that makeup the fluidic network. The mask may be a transparency that may becontacted with a photoresist, for example, SU-8 photoresist, to producea negative master of the photoresist on a silicon wafer. A positivereplica of PDMS may be made by molding the PDMS against the master, atechnique known to those skilled in the art. To complete the fluidicnetwork, a flat substrate, e.g., a glass slide, silicon wafer, or apolystyrene surface, may be placed against the PDMS surface and plasmabonded together, or may be fixed to the PDMS using an adhesive. To allowfor the introduction and receiving of fluids to and from the network,holes (for example 1 millimeter in diameter) may be formed in the PDMSby using an appropriately sized needle. To allow the fluidic network tocommunicate with a fluid source, tubing, for example of polyethylene,may be sealed in communication with the holes to form a fluidicconnection. To prevent leakage, the connection may be sealed with asealant or adhesive such as epoxy glue.

In some embodiments, the microfluidic networks described herein can becombined with one or more microfluidic components such as valves, pumps,droplet formation regions (e.g., in the form of a flow-focusing device),membranes, as well as those described in U.S. Application Ser. No.60/925,357, filed Apr. 19, 2007, and entitled “Manipulation of Fluidsand Reactions in Microfluidic Systems”, which is incorporated herein byreference in its entirety for all purposes. Any of a number of valvesand/or pumps, including peristaltic valves and/or pumps, suitable foruse in a fluidic network such as that described herein can be selectedby those of ordinary skill in the art including, but not limited to,those described in U.S. Pat. No. 6,767,194, “Valves and Pumps forMicrofluidic Systems and Methods for Making Microfluidic Systems”, andU.S. Pat. No. 6,793,753, “Method of Making a Microfabricated ElastomericValve,” which are incorporated herein by reference.

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

Example 1

This example shows the design, fabrication, and operation of amicrofluidic system for positioning, trapping and storing single cellsin chambers according to one embodiment of the invention.

An array of chambers was designed to position, trap and store cells, thechambers having a configuration such as the ones shown in FIG. 11.Chambers were designed to have a flow restriction region at one end ofthe chamber such that cells would be trapped when they flowed into thechambers. Once a cell was positioned in a chamber, this increased thehydrodynamic resistance of the chamber. This caused subsequent cells inthe fluid to enter a bypass channel extending from the chamber, insteadof the chamber itself. This method allowed the formation of an array ofsingle cells in the chambers.

In this example, the chambers were designed so they were just as wide asa single cell, with dimensions 5 μm high, 5 μm wide, and 400 μm long.Because of this restriction in height and width of the chamber, thecells were constrained to grow in a single line. While thecross-sections of the cells were approximately round, the chambers wereapproximately square, enabling media to be continually perfused throughthe chambers even while the cells were positioned in the chamber. Thevolume of a single chamber was 10,000 μm³ and there was approximately3,500 μm³ free volume when the chamber was filled with cells. Forlonger-term culture applications, a porous membrane can be incorporatedinto the device (e.g., a porous may form one surface of the chamber),which may allow for additional media exchange along the length of thechamber. Examples of semi-permeable membranes are described in moredetail in International Patent Apl. Serial No. PCT/US2006/034659, filedSep. 7, 2006, entitled “Microfluidic Manipulation of Fluids andReactions”, which is incorporated herein by reference in its entiretyfor all purposes.

Before the cells reached the chambers, they were passed through a filterthat was fabricated on-chip for both media and cell inlets. The smallestpore size of the filter was 5 μm, so aggregates of cells remainedtrapped in the filter. This filter helped to achieve the flow of onlysingle cells (instead of agglomerations of cells) into the chamberarray.

Over the time course of the experiments, cells trapped in the filtercontinued to grow. To avoid dislodged budding cells from the filter andcontaminating cell lineages in the chamber array, a flow-focusingjunction was included in the device design. During loading, media flowedin from the side channels (e.g., channels portions 1362 and 1364 of FIG.11) at a low flow rate and focused the stream of cells (e.g., flowingfrom channel portion 1360) so they entered the chamber array. Whenloading was complete, the flow of cells was stopped and the media flowrate was increased. While some of the media flowed into the chamberarray, media also flowed upstream into the channel containing the cells(e.g., channel portion 1360) and the filter. This upstream flow canprevent agglomerated and other cells trapped in the channel or filterfrom reaching the chambers.

Typical loading efficiencies of single cells in the chambers ranged fromabout 50-90%, e.g., depending on the flow conditions and the celldensity. The majority of the remaining chambers were empty, and in a fewinstances cases, contained multiple cells. The loading efficiencies canbe optimized by varying one or both of the flow rate and cell density.For example, in some cases, a loading flow rate of 95 μL/hr withapproximately 10⁷ cells/mL in the loading solution filled at least halfof the chambers with a single cell after approximately 5 minutes ofloading. In other cases, a concentration of approximately 10⁶ cells/mLwas used. Depending on the flow rate and other conditions, an increaseof the cell concentration or the loading time may lead to thepositioning of multiple cells per chamber.

In chambers of these dimensions used in this experiment, over 100 cellsderived from a single cell were captured in each chamber. Chambers mayalso be made longer to follow more generations. Over the course of1000-1500 minutes, a relatively constant division time was observed,even for cells positioned adjacent the flow constriction region. Thismay suggest that the division of cells was not limited by the number ofcells in the chamber (or by the dimensions of the chamber).

Once the cells are loaded in the chambers, a buffer was flowed throughthe chambers at rates of 55 μL/hr. Based on the volume of the device,the media was exchanged on timescales much less than the division timeof the cells.

To evaluate growth of cells in the progeny chambers, cells were trackedusing time-lapse microscopy. Images were acquired every 7 minutes. Theaverage cell division time was comparable to bulk growth rates.

The chambers facilitated fast and efficient qualitative screening ofsingle cells and their progeny. At the end of an experiment, images ofthe lines of cells were acquired. The number of single cells in “ON” and“OFF” states was easily determined by simple analysis of the proportionof light to dark channels. The chambers also made it easy to investigateswitching frequency for single cells.

For more detailed analysis of cells and their progeny, time-lapseimaging and analysis was performed. It was observed that cells and theirclosest progeny remained relatively close to each other within thechannel, however, recently budded cells sometimes squeezed past eachother in the chamber and disrupted the genealogical order of the cells.Furthermore, while the haploid cells typically budded from the same end,they occasionally reoriented in the channels and budded from the otherside. The cells and their lineology were tracked manually. However, thecells may also be tracked automatically, e.g., using software that canidentify individual cells (e.g., based on differences in fluorescenceintensity).

The chambers allowed for growth of cells to densities higher than thatwhich can normally be obtained in traditional laboratory conditions, forexample, up to 1×10¹⁰ cells/mL. This may facilitate studies of highdensity cell cultures, such as those found in nature.

With slight modifications in size, the progeny chambers may also be usedfor the culture of other suspension cells, such as mammalian blood cellsor stem cells. By treating the channels with appropriate surfacecoating(s) (e.g., fibronectin), adherent cells may also be grown in thechambers.

Device Fabrication.

The microfluidic device used in this example was fabricated as follows.The designs for the chambers were generated in AutoCad. Chrome maskswere printed on quartz (HTA Photomask, CA) and soft lithography was usedto create polydimethylsiloxane (PDMS) devices. In brief, a SU8 2005photoresist (MicroChem, Newton, Mass.) was spin-coated onto a siliconwafer (rinsed with methanol and prebaked for 10 minutes at 210° C.) to afinal thickness of 5 μm following the protocol described by themanufacturer. A mask was placed on top of the wafer and exposed to UVlight for 12 seconds. Exposure of the photoresist to UV light (OAI, SanJose, Calif.) crosslinked the exposed pattern, and the non-exposedphotoresist was dissolved away using propylene glycol monomethyl etheracetate (PGMEA). The channel height was confirmed to be 5 μm using ascanning profilometer (Stylus). PDMS was mixed with a crosslinker at aratio of 10:1, and poured onto the master (Sylgard 184 SiliconeElastomer, Dow Corning, Midland, Mich.). Devices were placed in a vacuumto rid the PDMS of air bubbles for at least 5 minutes before baking at65° C. overnight. A biopsy punch (0.75 mm diameter, Harris Uni-Core, TedPella Inc., Redding, Calif.) was used to punch entry and exit holes inthe PDMS. The PDMS was then oxygen plasma treated and bonded to a LabTekchamber with a no. 1.5 coverslip bottom (Nunc). The chambers were foundto be most stable during the course of scanning the stage and automatedimage acquisition.

Loading.

The microfluidic device was loaded with the yeast cells as follows.Yeast cells (S. cerevisiae) were cultured in YPD at 30° C. to a densityof OD600˜0.1 and were washed three times in SD media, and diluted100-fold. A dilute suspension of the yeast cells and perfusion mediawere loaded into 1 mL plastic syringes (BD, VWR). Needles (luer-lok, 27½gauge) were connected to the syringes, and the syringes were inverted toremove all air bubbles. The needles were then connected to PE-20 tubing(VWR/ALA Scientific Instruments Inc., Westbury, N.Y.). Syringe pumps(Kent/Harvard Apparatus) were used to control fluid flow. To begin, apiece of tubing was inserted into the exit port for waste collection,and the cell inlet was blocked with a plug. Plugs were fabricated byholding the tip of a 2″ long piece of PE-20 briefly in a flame, and thenplacing the melted end of the tubing between two flat objects. Thisformed a nice handle making the plug easy to hold. The remaining longend of the tubing was cut to make a short stub. With the plug insertedin the cell entry port, media was flowed through the media entry port ofthe device until the chambers were filled with fluid. The plug was thenreplaced with the cell tubing. Cells were loaded using flow rates of 95μL/hr cells and 15 μL/hr media. When about half of the chambers werefilled with single cells, the cell tubing was removed from the syringeneedle, and the media flow was increased to 45 μL/hr. This flow rate wasmaintained for the remainder of the experiment.

The cells were stained by connecting a media syringe to a three-waystopcock (Small Parts). A syringe full of fixation medium/stainingmedium was attached to the third entry of the stopcock at the end of agrowth experiment. The staining media was then flowed through thechambers. The chambers were imaged by placing the chambers on the stageof an inverted microscope (Zeiss Axiovert 200M). A timelapse series ofimages were acquired at different stage positions. Confocal microscopywas performed on a Zeiss microscope.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits; and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method of positioning a plurality of cells,comprising providing a microfluidic network comprising a microfluidicinlet, a first region, and a second region, wherein the first region andthe second region are in fluid communication; flowing a first fluidthrough the microfluidic inlet, the first region, and the second region,wherein the first fluid comprises a plurality of cells; and positioninga first cell in the first region, wherein said positioning preventsentry of another cell into the first region, wherein the first cell isencapsulated in a droplet or a gel.
 2. The method of claim 1, whereinthe first region has a lower hydrodynamic resistance than the secondregion prior to positioning the first cell.
 3. The method of claim 1,further comprising positioning a second cell in a third regiondownstream of the second region in the microfluidic network.
 4. Themethod of claim 1, wherein the microfluidic network comprises a fluidrestriction region between the first region and the second region. 5.The method of claim 4, wherein the first cell is prevented from passingthrough the fluid restriction region.
 6. The method of claim 4, whereinthe fluid restriction region is immediately adjacent to the firstregion.
 7. The method of claim 1, wherein at least a portion of thefirst region comprises a structure element to impart resistance forfluid flow into the first region.
 8. The method of claim 7, wherein thestructure element is selected from the group consisting of a valve, asemi-permeable plug, and a membrane.
 9. The method of claim 1, furthercomprising lysing the first cell in the droplet or the gel.
 10. Themethod of claim 9, further comprising labeling sequences released fromthe first lysed cell with a DNA probe.
 11. The method of claim 1,further comprising multiplying the first cell in the droplet or the gel.12. The method of claim 1, further comprising labeling the first cellwith an identifier.
 13. The method of claim 12, wherein the identifieris a dye, a probe, an antibody, or a fluorescent protein.
 14. The methodof claim 1, wherein the first cell is a yeast cell, a bacterium, amammalian cell.
 15. The method of claim 1, wherein the droplet or gelfurther comprises one or more reactants, nutrients, or drugs.
 16. Themethod of claim 1, further comprising conducting a reaction in thedroplet or gel.
 17. The method of claim 16, wherein the reaction is anamplification reaction.
 18. The method of claim 17, wherein the reactionis a polymerase chain reaction (PCR).
 19. The method of claim 1, furthercomprising analyzing a secreted component and/or growth rate of thefirst cell.
 20. The method of claim 1, further comprising growingcolonies from the first cell.
 21. The method of claim 1, furthercomprising flowing a second fluid through the microfluidic network, thefirst region, and the second region, wherein the second fluid comprisesa second reactive species.
 22. The method of claim 21, wherein thesecond reactive species interacts with the first cell.
 23. A method ofpositioning a plurality of cells, comprising providing a microfluidicnetwork comprising a microfluidic inlet, a first region, and a secondregion, wherein the first region and the second region are in fluidcommunication, and wherein at least a portion of the first regioncomprises a valve, a semi-permeable plug, or a membrane to impartresistance for fluid flow into the first region; flowing a first fluidthrough the microfluidic inlet, the first region, and the second region,wherein the first fluid comprises a plurality of cells; and positioninga first cell in the first region, wherein said positioning preventsentry of another cell into the first region.
 24. The method of claim 23,further comprising lysing the first cell in the first region.
 25. Themethod of claim 23, further comprising immobilizing the first cell to asubstrate.
 26. The method of claim 23, further comprising multiplyingthe first cell in the first region.
 27. The method of claim 23, furthercomprising labeling the first cell in the first region with anidentifier.
 28. The method of claim 27, wherein the identifier is a dye,a probe, an antibody, or a fluorescent protein.
 29. The method of claim23, wherein the first cell is a yeast cell, a bacterium, a mammaliancell.
 30. A method of positioning a plurality of cells, comprisingproviding a microfluidic network comprising a microfluidic inlet, amicrofluidic outlet, a chamber unit comprising a chamber inlet, achamber, a chamber outlet, a chamber bypass channel between the chamberinlet and chamber outlet, and a fluid restriction region fluidlyconnected between the chamber inlet and chamber outlet, wherein thechamber inlet is fluidly connected to the microfluidic inlet and thechamber outlet is fluidly connected to the microfluidic outlet; flowinga first fluid through the microfluidic inlet and the first chamber unit,wherein the first fluid comprises a plurality of cells; and positioninga first cell in the chamber of the first chamber unit, wherein saidpositioning causes another cell to flow through the chamber bypasschannel.
 31. The method of claim 30, further comprising lysing the firstcell in the first region.
 32. The method of claim 30, further comprisingimmobilizing the first cell to a substrate.
 33. The method of claim 30,further comprising multiplying the first cell in the first region. 34.The method of claim 30, further comprising labeling the first cell inthe first region with an identifier.
 35. The method of claim 34, whereinthe identifier is a dye, a probe, an antibody, or a fluorescent protein.36. The method of claim 30, wherein the first cell is a yeast cell, abacterium, a mammalian cell.